The AGREE instrument has been validated for evaluating Clinical Practice Guidelines (CPG) pertaining to medical care. This study evaluated the reliability and validity of physical therapists using the AGREE to assess quality of CPGs relevant to physical therapy practice.A total of 69 physical therapists participated and were classified as generalists, specialist or researchers. Pairs of appraisers within each category evaluated independently, a set of 6 CPG selected at random from a pool of 55 CPGs.Reliability between pairs of appraisers indicated low to high reliability depending on the domain and number of appraisers . The highest reliability was achieved for Rigour of Development, which exceeded ICC> 0.79, if scores from pairs of appraisers were pooled. Adding more than 3 appraisers did not consistently improve reliability. Appraiser type did not determine reliability scores. End-users, including study participants and a separate sample of 102 physical therapy students, found the AGREE useful to guide critical appraisal. The construct validity of the AGREE was supported in that expected differences on Rigour of Development domains were observed between expert panels versus those with no/uncertain expertise (differences of 10–21% p = 0.09–0.001). Factor analysis with varimax rotation, produced a 4-factor solution that was similar, although not in exact agreement with the AGREE Domains. Validity was also supported by the correlation observed  between Overall Assessment and the Rigour of Development domain.These findings suggest that the AGREE instrument is reliable and valid when used by physiotherapists to assess the quality of CPG pertaining to physical therapy health services. Clinical practice guidelines (CPGs) are one option for promotion of quality in health services -6 Many cThe AGREE Collaboration published the development process and associated reliability and validity data in 2003 This repThe AGREE Collaboration also assessed face, construct and criterion validity of the AGREE Instrument. Face validity was determined by surveying appraisers attitudes and opinions about the instrument and its associated user guide. Construct validity was determined by comparing scores of guidelines in different subgroups to determine whether they fit three specific constructs. The constructs tested included whether established quality guideline programs produced guidelines with higher domain scores than those developed outside of established systems; whether guidelines supported by well-documented technical information had higher domain scores than those without such documentation; and finally whether guidelines developed as national policies were higher quality than regional or local CPGs. The first hypothesis was supported with respect to editorial independence, but not other domains. The second and third hypotheses were supported with respect to the domain Rigor of Development, but not other domains. Finally, criterion validity was determined by assessing the rank correlation between appraisers domain scores and their overall assessment scores . These correlations were all highly significant .Physical therapy and other health care disciplines shares common challenges in providing effective care within limited resources. While many disciplines currently use the AGREE the validation paper emphasized medical practice and practitioners. The nature of physical therapy practice differs substantively from medical practice in a number of ways including access, funding, the nature of interventions, research systems and professional associations; all of these might affect the type of CPGs, developed. Differences in training between disciplines might also cause variations in how the AGREE was interpreted. Additional validation with other settings or users would strengthen the validation of the AGREE across a variety of applications.CPGs have arisen within the field of physical therapy from a variety of sources,16 ProfeThis study was a cross-sectional study conducted to evaluate inter-appraiser reliability and validity of quality appraisal of CPGs performed by physical therapists using the AGREE instrument Permission to use the AGREE instrument was obtained from the AGREE Collaboration. Ethics approval for this study was received from the University of Toronto, Office of Research Services.Clinical practice guidelines (CPGs) are "systematically developed statements to assist practitioner and patient decisions about appropriate healthcare for specific clinical circumstances". The CPGAll participants were physical therapists who were recruited through advertisements in professional newsletters. A total of 72 therapists responded to advertisements and agreed to participate, 69 actually participated in training and study evaluations – two others had personal circumstances that prevented them from attending training and one failed to respond further. The participants were classified according to the following criteria: 1. Clinical Specialists were Physical Therapists who were currently practicing in a specific area, had a minimum of three years experience and had participated in at least one post-graduate course per year in their area of clinical expertise. 2. Generalists were Physical Therapists practicing in a variety of areas of physical therapy or an ongoing general practice, which covered a broad-spectrum of neurological, orthopaedic and cardiorespiratory health problems. 3. Researchers were Physical Therapists with or approaching completion of a graduate research degree (M.Sc. and/or PhD) with experience in conducting clinical trials or outcomes research and experience in formal critical appraisal of clinical research.Demographic data was collected on all participants. Eligible participants were provided with training materials which included the AGREE instrument (form and associated interpretation guidelines),18 as weSubsequently, all participants met by teleconference for one hour with a subgroup (4–8) of study participants. The sessions  were led by a single facilitator (first author). During this session participants reviewed their responses to each question and discussed their rationale or concerns regarding scoring. The facilitator did not indicate a correct score for any individual item. Participants were instructed that the group facilitator would not indicate whether a given score was correct, as this was not possible for many of the items. Participants were directed to appreciate the difference between items that had clear answers because they inquired about specific factual information versus those that had a subjective element where responses might vary according to context. Participants were corrected if they incorrectly interpreted the meaning of a given item on the AGREE instrument. Although consensus was not the objective of the training sessions, participants tended to come to consensus after discussing items with colleagues and a facilitator. In total, 69 appraisers attended the training sessions.The appraisal instrument used to evaluate the CPGs was the AGREE . This inScope And Purpose: 3 items that address the overall aim of the guideline, the clinical question and the target population1. Stakeholder Involvement: 4 items that address the composition, expertise and representation of the development group2. Rigor Of Development: 7 items that evaluate the process used to locate and synthesize the evidence and to formulate and update the recommendations,3. Clarity And Presentation: 4 items that address language and format4. Applicability: 3 items that address the potential organizational, behavioural and cost implications of implementation and5. Editorial Independence: 2 items that address potential conflicts of interest.6. Items are rated on a 4-point scale with endpoints of 4 'strongly agree' and 1 'strongly disagree'; the two midpoints are 3 'agree' and 2 'disagree'. A section for overall assessment is included at the end of the instrument that requires the appraiser to make a judgment about the overall quality of the CPG. Appraisers are asked whether they would 'strongly recommend', 'recommend ', 'would not recommend' or are 'unsure' if they would recommend the CPG for practice.All participants completed the training program and proceeded to evaluate a set of 6 CPGs. These CPGs, six copies of the AGREE instrument, and a pre-paid return were provided by mail. Each guideline was evaluated independently by three pairs of appraisers who were randomly picked from the three pools of Physical Therapists . All appraisers returned their packages, although up to 3 reminders calls were required for late returns. Participants who completed the study were provided with an honorarium ($100).Data analysis was conducted to verify the quality of data, assess instrument reliability and determine the validity of the AGREE instrument for physical therapy practice. SPSS statistical software for Windows  was used for all statistical analyses. P-values of 0.05 or less were considered significant.Data entry was completed by a single research assistant who inspected data for errors once the data file was complete. The first author conducted random checks of data entry against original data sheets. Descriptive statistics were conducted to identify outliers or unusual values.Domain scores of each CPG were calculated as recommended by the AGREE Collaboration. The scores of the individual items in the domain were summed and standardized as a percentage of the maximum possible score for that domain (AGREE Collaboration, 2001).The internal consistency of each domain was evaluated using Cronbach's Alpha. The reliability between appraisers was determined for each question and each domain of the AGREE. Intraclass correlation coefficients (ICC 1,1) were calculated within each pair of appraisers and across the pool of appraisers. A one-way random effects model was used as pairs of appraisers were randomly selected from our pool of physical therapist appraisers. An unweighted and quadratic weighted kappa were calculated to indicate the agreement within pairs of appraisers on whether a CPG was appropriate for clinical utilization.ICCs or kappa values above 0.75 were considered to represent good, 0.40–0.75 moderate and <0.40 poor reliabilityThe face validity of the instrument for physical therapy practice was determined from feedback provided on the instrument from two sources. Participants  in the study were invited to provide feedback at the training sessions . They were also asked to add comments about any items, any issues with clarity or concerns directly on their AGREE form when they were using the AGREE on their assigned CPGs. These were returned, by mail, with their ratings. In addition, over the course of two years, a sample of 102 entry-level-masters trainees at McMaster University were provided the training materials (except for multiple-choice questionnaire) and were required to complete an assignment where they evaluated the same CPG used durThe validity of the domain structure was evaluated using a principal components, varimax rotated factor analysis. Item means across all 6 appraisers were entered into the analysis. Coefficients were evaluated to determine whether they supported the domain structure and followed a similar pattern as to that reported for a previous factor analysis published by the AGREE Collaboration.Construct validity was assessed by evaluating 2 hypotheses. The first hypothesis was selected to match the hypothesis tested by the AGREE Collaboration guidelines and suppFinally, criterion validity was assessed in the same manner as reported by the AGREE Collaboration in their validation study. Again we recognize we did not have an external criterion. Kendall Tau B Rank correlation coefficients were calculated between the appraisers domain scores and the overall assessment score.Sixty-nine physical therapists were recruited and were categorized as clinical specialists (n = 29), generalists n = 21) or researchers (n = 19) , followed by neurological (24%) and cardiorespiratory (10%). One participant reported to specialize in paediatric physical therapy. Two participants did not specify their area of specialization.Analysis of reliability of individual items indicates a trend for higher reliability in items within the domain from Rigor of Development  uniformly agreed that the AGREE instrument provided a useful structure and guidance in the evaluation of the CPG. Students compared the AGREE instrument to evaluation instruments they had used for critical appraisal of different study designs, such as clinical trials and systematic reviews. Students frequently commented that these previous instruments had a more open-ended format and expected the reviewer to understand issues in critical appraisal with little direction as to expectations or scoring criteria. Thus, they found the concrete nature of the AGREE instrument and the clear instruction on interpretation to be a useful framework for the evaluation process. Students stated that this direction increased their confidence that they had addressed all important issues. Although students differed on their ratings for individual questions, as well as the overall usefulness of the CPG evaluated, the majority of students understood and correctly interpreted the intent of the items from all of the AGREE domains. The majority of the students, 96 %, stated that they would use the AGREE instrument in other situations. A concern raised by the remaining 4% and other students who would continue to use the instrument was the length of the form and the amount of time required to complete the evaluation, given the busy nature of clinical practice.The factor analysis supported a 4-factor solution. The first factor explained 45% of the variance and contained items primarily from the Scope and Purpose or Rigor of Development domains. The second component explained 12% of the variance and contained items primarily from the Clarity and Presentation or Applicability domains. The third factor explained 7.7% of the variance and contained all of the items from the Stakeholder Involvement domain, all of the Editorial Independence items and question 13 from Rigor of Development, which pertains to whether the guideline has been externally reviewed by experts. The fourth component explained 5.6 percent of the variance and contained item 11 regarding health benefits/side effects and item 15 regarding whether recommendations were specific and unambiguous. Item means and their loadings are presented in Table The construct that the CPGs developed by expert guideline development groups would have a higher score on the domain Rigor of Development was supported  to moderate (0.64), with the highest correlation being observed for the Rigor Of Development domain.  and whether key review criteria for monitoring or audit had been provided (2/8 ICCs were not greater than 0). Study participants also appeared to have difficulty interpreting these items during the training session and thus these disagreements may reflect a lack of consensus about the meaning of these items. In some CPGs information on Editorial Independence is contained in footnotes and was missed by some evaluators. Improvements in reliability on these items might be anticipated with further training. Other items where low reliability was observed included whether the clinical question was specifically described (6/8 ICCs were not greater than 0) and whether the patients to whom the guideline applied were specifically described (2/8 ICCs were not greater than 0). During training, participants appeared to understand these questions, although expectations about what constituted an appropriate description of the clinical question or patient population tended to vary according to the participant's level of expertise in that given clinical area. Thus, disagreements on these items may partially relate to differences in priorities or familiarity with relevant issues between participants. Further training is unlikely to enhance reliability in this case, but raises the question about the importance and specificity of relevant clinical expertise when selecting evaluators. Although our intent was to evaluate the importance of clinical expertise, we classified participants quite broadly. Participants classified as clinical specialists were provided CPGs within their broad area of practice; but, we did not ascertain whether in fact the specific topic of the CPG was an area in which they actually did have knowledge or experience. For example, a clinician with expertise in musculoskeletal practice might practice primarily in a narrower area within the field, such as, rheumatology, upper extremity, lower extremity, joint replacements etc. In such cases, their familiarity with the salient features of the specific conditions might be less detailed and result in a differential evaluation regarding whether the clinical population had been appropriately defined. Future investigations should focus on content knowledge in a more specific sense to determine the importance of this issue when selecting evaluators.It is not uncommon for the policy developers to use reliability data to set standards for the number of appraisers required when establishing guideline evaluation processes. Our data provide little direction in this regard, except to suggest that more than two appraisers are advisable. This is consistent with the minimum recommendation of 2 made by the AGREE Collaboration.  MeasuremAlthough interpretation of validity analyses is complex and requires some subjective decisions, overall our validity analyses are supportive of the AGREE instrument and are substantively similar to that reported by the AGREE Collaboration for 33 medically based CPGs A factorConstruct validity tests also supported the validity of the AGREE instrument. Constructs were developed to replicate those used in the previous validation of the AGREE and provOverall, our findings were remarkably consistent with those reported by the AGREE collaboration when validating the AGREE as used by medical practitioners Validati1. The AGREE instrument is reliable and valid when used by physical therapists to assess the quality of clinical practice guidelines2. A minimum of three appraisers should be used to optimise reliability, although issues on effecting knowledge transfer or maximizing validity might increase these requirements.3. There is no evidence that specific types of physical therapists provide more reliable scores. As this study did not address whether familiarity with the actual content of the CPG influenced reliability, this should be studied in the future.4. There is evidence that guidelines developed with the assistance of experienced guideline developers are more rigorous.5. The AGREE instrument was found to be useful in assisting both novice and experienced therapists in evaluation of clinical practice guidelines. The majority of therapists would continue to use the instrument.6. Validity analysis supported the majority of results reported by the AGREE Collaboration) The typeIG is an associate member of the AGREE Collaboration.JM, DB, SS, SSW formed the original study team which conceived of the research question, obtained the ethics approval and obtained grant funding. JM developed the research design, conducted statistical analyses, developed and facilitated the training sessions, developed and evaluated the student evaluations and drafted the manuscript. DB managed grant funds, selection of clinical practice guidelines, appraiser recruitment and data collection. All authors approved the final study protocol, contributed to interpretation of the study results and participated in revisions of the manuscript. IG provided consultation with respect to CPG research and health care policy. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:The training testClick here for file


The purpose of this study was to characterize thumb motor dysfunction resulting from simulated lower median nerve lesions at the wrist.Bupivacaine hydrochloride was injected into the carpal tunnel of six healthy subjects to locally anesthetize the median nerve. Motor function was subsequently evaluated by measuring maximal force production in all directions within the transverse plane perpendicular to the longitudinal axis of the thumb. Force envelopes were constructed using these measured multidirectional forces.Blockage of the median nerve resulted in decreased force magnitudes and thus smaller force envelopes. The average force decrease around the force envelope was 27.9%. A maximum decrease of 42.4% occurred in a direction combining abduction and slight flexion, while a minimum decrease of 10.5% occurred in a direction combining adduction and slight flexion. Relative decreases in adduction, extension, abduction, and flexion were 17.3%, 21.2%, 41.2% and 33.5%, respectively. Areas enclosed by pre- and post-block force envelopes were 20628 ± 7747 N.N, and 10700 ± 4474 N.N, respectively, representing an average decrease of 48.1%. Relative decreases in the adduction, extension, abduction, and flexion quadrant areas were 31.5%, 42.3%, 60.9%, and 52.3%, respectively.Lower median nerve lesion, simulated by a nerve block at the wrist, compromise normal motor function of the thumb. A median nerve block results in force deficits in all directions, with the most severe impairment in abduction and flexion. From our results, such a means of motor function assessment can potentially be applied to functionally evaluate peripheral neuropathies. The thumb has unique anatomical and biomechanical characteristics that are required to perform many manipulative tasks. Thumb motor dysfunction resulting from neuromuscular and musculoskeletal pathologies severely hinders the performance of these daily tasks. Clinical treatment, prevention protocols, and rehabilitation efficacy requires a thorough understanding of thumb motor capabilities, as well as its associated functional deficit. Investigations of underlying pathological mechanism of the thumb help advance clinical treatments such as tendon transfers , functioMeasurement of strength during maximum voluntary contraction is a simple and direct means of assessing neuromuscular function. Popular instruments used for quantitative assessment of thumb strength are pinch dynamometers. The pinch output, however, provides limited information about thumb motor function in that it offers a single generic force in one specific direction. Each muscle/tendon within the thumb has a distinct anatomical origin and insertion, suggesting its external force potential in a particular direction -6. HenceDisorders resulting from traumatic injuries to and various diseases of these nerves are common in clinical practice. Clinical manifestations of hand dysfunction are distinctive depending on the nerve involved. For example, thenar atrophy is a major clinical observation affecting thumb function at the later stages of compression neuropathy of the median nerve. Several studies have been conducted to investigate the effects of simulated peripheral neuropathies using local anesthetization ,16,20,21The purpose of this study was to utilize our developed apparatus and protocols to investigate the effects of lower median nerve lesion on thumb motor function. The lesion was simulated by blocking the median nerve at the wrist using an anesthetic. We hypothesized that a median nerve block would cause (1) a decrease in force production, which would be direction-dependent with the most severe reduction in the abduction direction, and (2) a decrease in the force envelope area and force quadrant area, with the greatest decrease in the abduction quadrant.Six healthy male subjects (mean age: 26.9 ± 5.1 years) participated in this study. The subjects had no previous history of neuromuscular or musculoskeletal disorders of the upper extremities. Each subject signed an informed consent form approved by the Institutional Review Board prior to participating in the experiment.Injections were performed under aseptic conditions while the subjects sat with the forearm supinated and the wrist slightly extended. After the skin at the palmer area of the wrist was cleaned with alcohol, 4 mL of 0.5% bupivacaine hydrochloride  was injected into the carpal tunnel with a sterile 25-gauge short-bevel needle. The needle was inserted through the transverse carpal ligament in line with the radial border of the fourth digit slightly ulnar to the palmaris longus tendon at the level of the distal wrist crease. Forty minutes was allowed for the median nerve block to reach complete effectiveness  and was The experimental apparatus was designed and constructed to measure maximum voluntary contraction forces of any digit at any point along the digit. Force application was possible in any direction within the transverse plane of the longitudinal axis of the digit. The apparatus consisted of position control accessories, a force transducer, and a custom fitted aluminum ring attached to the transducer Figure . The traThe analog outputs from the transducer were digitized using a 16-bit analog-to-digital converter . The X (abduction/adduction) and Y (flexion/extension) force components in the transverse plane were displayed on the screen while the subject performed a force production task. The resolutions of the force transducer in its axial (flexion/extension) and horizontal (abduction/adduction) directions were 0.16 N and 0.08 N, respectively. A personal computer equipped with LabVIEW  was used for force data acquisition, display, and processing.Each subject was tested before and after median nerve block. The nerve block procedures were performed immediately after the completion of the first testing session. Post-block testing started after the verification of complete median nerve block, approximately 40 minutes after the injection. During each test, the subject was seated in a chair adjacent to the testing station modified with a wooden board to align their back vertically throughout the trials. The subjects rested their forearm on padded wooden blocks positioning their shoulder in approximately 60° of frontal plane abduction. Nylon straps fitted with plastic snap locking mechanisms secured the forearm and minimized the intervention of the elbow and shoulder during thumb force application. Subjects grasped a vertical dowel secured to the distal end of the wooden blocks in a midprone position. Formable thermoplastic braces were used to fix the elbow in 90° of flexion, and the wrist in 20° of extension and 0° of ulnar deviation. A metallic brace was used to fix the interphalangeal joint of the thumb in full extension. The aluminum ring was placed around the middle of the proximal phalanx and oriented to accommodate comfortable thumb position with the metacarpophalangeal joint flexed approximately 15°. Prior to testing, a line was drawn on the proximal phalange at the midpoint between the interphalangeal and metacarpophalangeal joints. The alignment of the ring with the circumferential line standardized the location of force application within and between subjects. As force application was at the middle of the proximal phalanx, mechanical action pertains to both the metacarpophalangeal and carpometacarpal joints. We chose the terminology of flexion/extension and adduction/adduction based on the mechanical action with respect to the metacarpophalangeal joint. With the thumb in the ring Figure , extensiEach subject performed 15 circumferential MVC trials with randomized starting directions Figure . The subData from multiple circumferential trials were accumulated to construct a force envelope. The procedures to generate a force envelope were as follows:Xi, Yi) were transformed into polar coordinates (Rα, α), where Rα was the force magnitude at an angular position α. Each α was rounded to the nearest integer ranging from 0 to 359 degrees.Cartesian force coordinates  to obtain filtered maximal forces. These forces formed a force envelope.A moving average with an interval of 10° was applied to the maximal series data quadrant force was represented using the mean magnitude of the forces in that quadrant. The areas of the entire envelope and each quadrant were calculated by summing the areas of individual arc sections formed by the polar coordinates of the force envelope.  were used to analyze outcome measures. The independent variables were testing SESSION (n = 2, i.e., pre- and post-block), force DIRECTION (n = 16), and force QUADRANT (n = 4), with SESSION as a repeated variable. Dependent variables were directional force, individual quadrant area and force envelope area. Statistical analyses were performed using SPSS 11 (SPSS Inc., Illinois) with statistical significance set at α = 0.05.Figure A single force in each quadrant was represented using the mean magnitude of the forces in that quadrant (see description in the Methods). The average quadrant forces were significantly decreased after nerve block  were smaller than those reported in the literature. Kozin et al.  reportedAn unexpected finding from this study was that the force deficit occurred in all directions Figure . In otheThe median innervated muscles are the dominant abductors of the thumb metacarpophalangeal and carpometacarpal joint. The more than 50% residual abduction force found in this study suggests that the injection did not totally block the motor function of these muscles, even though a complete sensory loss was verified. This concurs with clinical observations of median compression neuropathy. Individuals with carpal tunnel syndrome complain of sensory dysfunction early in the disease process (at the beginning), while motor signs of thenar wasting and thumb weakness occur as the disease advances. The concept that the motor deficit is more resistant to peripheral median neuropathy than sensory loss has been well documented ,26,27. BIn conclusion, we have incorporated a method for assessing thumb motor deficit based on strength measurement with a standard local anesthetic to investigate the effects of a simulated median neuropathy on thumb motor function. Median nerve block results in force deficits in all directions, with the most severe impairment in abduction and flexion. Future endeavors using this methodology can potentially further elucidate underlying pathomechanisms of peripheral neuropathies in all digits of the hand.


Dizziness is a common complaint among the elderly with a prevalence of over 30% in people over the age of 65. Although it is a common problem the assessment and management of dizziness in the elderly is challenging for family physicians. There is little published research which assesses the quality of dizziness assessment and management by family physicians.We conducted a retrospective, chart audit study of patients with dizziness attending the Sunnybrook Family Practice Center of Sunnybrook and Women's College Health Sciences Center (SWCHSC) in Toronto. We audited a random sample of 50 charts of patients from 310 eligible charts. Quality indicators across all dizziness subtypes were assessed. These quality indicators included: onset and course of symptoms; symptoms in patients' own words; number of medications used; postural blood pressure changes; symptoms of depression or anxiety; falls; syncope; diagnosis; outcome; specialty referrals. Quality indicators specific to each dizziness subtype were also audited.310 charts satisfied inclusion criteria with 20 charts excluded and 50 charts were randomly generated. Documentation of key quality indicators in the management of dizziness was sub-optimal. Charts documenting patients' dizziness symptoms in their own words were more likely to have a clinical diagnosis compared to charts without (P = 0.002).Documentation of selected key quality indicators could be improved, especially that of patients' symptoms in their own words. Dizziness is a common complaint among the elderly, with a prevalence of more than 30% in people over age 65  and it aLife-threatening illness is rare in patients with dizziness (with cerebrovascular disease accounting for 6%, cardiac arrythmia for 1.5% and brain tumor for <1%) . HoweverWhen assessing dizziness, what concerns a family physician most are: 1) how to distinguish serious causes of dizziness from less urgent ones; 2) how to manage patients with chronic but yet debilitating dizziness; and 3) how to decide on the right timing and the appropriate specialty for referral. However, many family doctors describe dizziness as "confusing" and "discouraging" problem  and expeTraditionally, the approach to dizziness is "disease-oriented", in which the clinician aims, at a minimum, to exclude potentially fatal causes and possibly to diagnose a specific cause for treatment. On the other hand, some authors like Tinetti et al  and Kao Despite differences in the above two approaches, both share in common certain key quality indicators as reflected in recent studies and reviews ,7,11-15.st 2001 and Jan 31st 2003; 3) Patients 65 years of age or older when seen. Exclusion criteria were: 1) Patients who are discharged from service or died; 2) Patients whose presenting symptoms were not dizziness or any of its subtypes.A retrospective chart audit was conducted at the Family Practice Center (Sunnybrook Campus) of Sunnybrook and Women's College Hospital Health Sciences Center. Inclusion criteria for the chart audit were: 1) Patients with a International Classification of Disease (ICD-9) diagnostic billing code of "780" (dizziness); 2) Patients seen between Feb 1A chart audit intake form was designed , which iThe general outcome measures/quality indicators across all dizziness subtypes include the documentation of : 1) onset and course of symptoms; 2) symptoms in the patients' own words; 3) number of medications used; 4) postural blood pressure changes; 5) symptoms of depression or anxiety; 6) falls; 7) syncope; 8) diagnosis; 9) outcome of dizziness; 10) specialty referrals.The quality indicators specific to vertigo include the documentation of 1) episode duration; 2) relationship to head turning; 3) tinnitus and hearing loss; 4) ear examination; 5) neurological examination; 6) spontaneous nystagmus; 7) positional nystagmus  (16). These patients were also audited for whether audiometry was ordered and whether Epley's manoevre  was offeThe quality indicators specific to lightheadedness include the documentation of 1) relationship to postural change; 2) cardiac symptoms; 3) syncope; 4) orthostatic blood pressure changes. These patients were also audited for whether ECG or Holter monitoring were ordered.The quality indicators specfic to disequilibrium include the documentation of1) falls; 2) neurological exam; 3) cerebellar signs; 4) Gait examination; 5) Romberg's sign; 6) visual acuity.The quality indicators specifically to other non-classifiable dizziness include the documentation of symptoms of depression and anxiety.310 charts satisfied the inclusion criteria with 20 charts excluded. A random sample of 50 charts were generated for the audit. The demographics of the sample, including age, gender living situation, are described in Table The distribution of different subtypes of dizziness is described in Table The onset and diagnoses of dizziness are described in Table The documentation of general and dizziness subtype-specific quality indicators in history and physical examination are described in Table As for the course of dizziness, only 2 patients have worsening symptoms (4%) and 60% of patients are referred to specialty services, the commonest ones of which are ENT (12%), neurology (8%) and cardiology (6%).A striking finding from this study was that 46% of the patients did not have any diagnosis and 10% of them had more than one diagnosis. This finding is in accordance with the data from the review by Sloan et al  and illuEffective history taking and communication between family physicians and patients is of crucial importance in the diagnosis of dizziness. The present chart audit study showed that family physicians were more likely to reach a diagnosis when patients' symptoms were documented in their own words, compared to those without such documentation.Overall, the documentation rate of key quality indicators important to all dizziness subtypes were low, such as falls, syncope, symptoms of depression and anxiety, and orthostatic blood pressure changes. A history of falls is associated with increased morbidity but this was documented in only 36% of patients. This is especially worrying given that 28% of patients in our sample were known to be living alone. The finding that 60% of patients with dizziness are using 5 or more medications is consistent with Tinetti's population-based cross-sectional study .Among vertiginous patients, the documentation rates for episode duration, relationship to head turning, hearing loss, and Hallpike maneuver were far from satisfactory. Among lightheaded patients, the documentation rates for symptom relationship to postural change, chest pain, palpitation, syncope, and orthostatic blood pressure changes are better but there is still room for improvement. Among patients with disequilibrium, the documentation rates for falls, gait examination, cerebellar signs, Romberg's sign and visual acuity examination were again sub-optimal. Among patients with non-specific dizziness, symptoms of depression and anxiety were also sub-optimally documented.This study has several limitations. First, being a retrospective study, its strength is limited because there is no standardized strategy or protocol for data collection among different family physicians. Second, a chart audit is prone to documentation bias and incompleteness, depending on individual family physicians. In addition, the sample size of 50 is relatively small given the complexity of the clinical problem. Moreover, only one diagnostic billing code was used. Although this would catch the presenting symptom at its undifferentiated stage, the drawback is that we may underestimate the actual scope of the problem by ignoring patients who were coded more specifically by their dizziness subtypes. In addition, only one hospital site, namely Sunnybrook hospital, was selected for chart audit. Being an academic teaching center with predominantly older patients, the patient data from this hospital alone may not be generalizable to those of a community clinic setting.Future directions for study would include the conduction of more prospective cohort studies on primary care patients using a standardized protocol for data collection This would assure uniform and consistent evaluation with the least amount of selection bias. Ideally, the use of inception cohorts would allow for better definition of the causes and natural history of dizziness in persons having their first episode. More prospective studies on dizziness outcomes with extended follow-up periods, as well as studies on different management strategies and specialty referral patterns for dizziness in the primary care setting would be helpful to family physicians. The ultimate goal is to identify the clinical and demographic or situational characteristics in a primary care setting that could help predict management decisions, such as diagnosing the most likely attributable cause of dizziness, delivering the most effective treatment for the symptom, and making the most timely and appropriate referral.Regardless of whether one regards dizziness as a "geriatric syndrome" or as a discrete "disease" for which a clear assessment algorithm could be established, there are certain quality indicators that are helpful for either approach but their documentation were found to be sub-optimal in this chart audit study. Allowing patients to describe their symptoms in their own words may help to improve diagnosis and management.The author(s) declare that they have no competing interests.EK contributed to the design and conception of the study, carried out the chart audit, analyzed the data and wrote the first draft of the manuscript. NP contributed to the design and conception of the study, supervised the conduct of the chart audit and data analysis and wrote and revised subsequent versions of the manuscript.The pre-publication history for this paper can be accessed here:1. Chart Audit Form MS Word document with the chart audit form used in the studyClick here for file


Mycobacterium tuberculosis contain a majority of bacteria that can grow in broth cultures but cannot grow on solid medium plates. These may be in a non-replicating, dormant growth phase. We hypothesised that a similar population might be present in chronic, murine tuberculosis.Old, stationary cultures of M. tuberculosis, strain H37Rv, in the spleens and lungs of mice in a 7-day acute infection and in a 10-month chronic infection were made by conventional plate counts and, as broth counts, by noting presence or absence of growth in serial replicate dilutions in liquid medium.Estimates of the numbers of viable 10 cfu /organ from spleens and 5.523 log10 cfu/organ from lungs, while plate counts were 3.858 log10 cfu/organ from spleens and 3.662 log10 cfu/organ from lungs, indicating that the total bacterial population contained only 3.7% bacilli in spleens and 1.4% bacilli in lungs, capable of growth on plates.Plate and broth counts in 6 mice gave similar mean values in the acute infection, 7 days after infection. However, the broth counts were much higher in 36 mice with a chronic infection at 10 months. Broth counts averaged 5.290 logThe proportion growing on plates might be a measure of the "dormancy" of the bacilli equally applicable to cultural and animal models. Mycobacterium tuberculosis all grow well on plates and are estimated as colony forming units (cfu). However, cultures that have been grown undisturbed in the depths of liquid medium for 100 days contain a majority population which grows in liquid medium but is not able to form colonies on solid medium [3H] uridine of only 15% of log phase cultures [The organisms in a log phase, actively multiplying culture of d medium . Since td medium H uridineM. tuberculosis suspended in 0.1% gelatin. The spleens and lungs were obtained at sterile autopsy and were homogenised as described elsewhere [M. tuberculosis, namely a clear supernatant in undisturbed cultures with an upwards swirl of white growth on shaking,. Probable numbers of bacilli (pnb) per organ were obtained from a table of densities of organisms estimated by the dilution method [The media used were 7H9 liquid medium with 10% albumin, dextrose, catalase supplement and 0.05% Tween 80, and 7H11 agar medium with 10% oleic acid, albumin, dextrose, catalase supplement (Becton Dickinson, Oxford, UK). They were made selective by the addition of 100 μg carbenicillin, 200 U polymyxin B, 20 μg trimethoprim and 10 μg amphotericin B per ml (Mast, Bootle, UK) . The twon method . Samples6 cfu of the H37Rv strain. Plate and broth counts were carried out 7 days later.Each of 6 mice was infected by the intravenous route with 200 μl of a suspension of a containing 2.6 × 103 cfu of the H37Rv strain. Mice in our experiments are usually housed in an isolator, connected by a tunnel port to a Class 1 safety cabinet through which air from the environment is sucked. The intra-peritoneal route was chosen so that mice could be kept throughout the experiment in the isolator to prevent cross infection with mouse pathogens during exposure to the outside air in the Class 1 cabinet. One day after infection, samples of 6 mice yielded scanty or no colonies in plate counts of spleens and lungs. After a further 4 weeks these organ counts in 6 mice had risen to 2.24 × 104 in spleens and 1.15 × 104 in lungs. The mice were then divided into 6 experimental groups, 4 of which were vaccinated with various DNA vaccines over a 4-week period and 2 were unvaccinated controls. At 12 weeks after infection only 9 of 36 lungs and 18 of 36 spleens yielded positive growth on plates. At 10 months after infection, the 36 remaining mice were sacrificed, and plate and broth counts were set up on all, using dilutions estimated from a sample of 4 mice sacrificed 3 weeks earlier.Each of 88 mice was infected by the intra-peritoneal route with 200 μl of a suspension containing 3.1 × 10The results of the plate and broth counts were examined by 2-way analysis of variance using the Stata package, release 8 (Stata Corp., College Station, TX)-4 or 10-5 dilutions) of any set of broth cultures. The results in the 6 mice in the acute infection experiment and in 27 of the 36 mice in the chronic infection experiment that had assessable numbers of bacilli estimated as cfu by the plate method and as pnb by the broth method are set out in Table 10 cfu / organ and the plate count was 3.858 log10 cfu /spleen. The difference between these counts is 1.432 log10 cfu / spleen (27-fold) so that, on the assumption that all bacilli that grew on plates also grew in broth, the bacilli capable of growing on plates comprised 3.7% (100/27) of the total count. In the lungs the mean broth count was 5.523 log10 cfu /lungs, about 73-fold higher than the plate count for the lungs. Thus the bacilli able to grow on plates comprised about 1.4% (100/73) of the total. The differences between individual mice were significant (p = 0.01) and highly significant between the counting methods (p < 0.001).As the experimental vaccines appeared to have only small effects, which will be reported elsewhere, in the chronic infection model, the results in all 36 mice at 10 months are considered together. A typical broth count is shown diagrammatically in Table 10 cfu / ml) were 19-fold higher than the corresponding plate counts , in approximate agreement with the 27-fold (3.7%) estimate of the difference between broth and plate counts obtained from Table In the remaining 9 mice no colonies were obtained on the neat dilution plates in either the lungs alone or in both lungs and spleen Table . However10 0.8, that is about 12%, in the count, it is evident that counts increased during incubation, rapidly between 3 and 6 weeks and slowly between 6 and 9 weeks.The following changes occurred in the broth counts during incubation. The counts between the 3-week and the 6-week readings increased on average in the lungs of each acute mice by 2.3 tubes and by 4.7 tubes in the spleens, and in each of the chronic mice by 2.0 tubes in lungs and 1.6 tubes in spleens. Thereafter, the increase from 6 weeks to 9 weeks was 1.5 tubes in the lungs of acute mice and 0.83 tubes in their spleens, while the increases in the lungs of chronic mice were 1.3 tubes and 1.0 tube in the spleens. Since an increase of 1 tube indicates a rise of about logM. tuberculosis was provided by the growth of typical colonies on 7H11 plates. That it was not due to sporadic contamination was shown by the usual complete absence of contamination in selective media, by a clear supernatant in the unshaken cultures and by the absence of any growth in the "no growth" zones of the broth cultures.The chronic infection experiment was unusual in that the intraperitoneal infection in vaccinated mice led to trapping of the bacilli in the peritoneal cavity, so that few bacilli reached the organs, and thus the plate counts were sometimes negative, with a count of less than 25 cfu / organ. Variation, considerably greater than after intravenous or airborne infection, was also evident, the SD of the 4-week spleen counts, expressed as log10 cfu/organ, being 0.51 as compared to 0.23 for intravenous infection [10 pnb / ml, while a smaller population B, that grew on plates, was estimated from parallel plate counts as 5.85 log10 cfu / ml.[In log phase cultures  and in the acute infection of mice, similar estimates of viable organisms were obtained in plate and broth counts However, our best estimate indicated that the bacilli in the chronic infections that would grow on plates was about 3.7% of the total population in spleens and 1.4% in lungs. This can be compared to the findings on a culture in 7H9 broth grown undisturbed for 100 days in the depths of liquid medium with caps screwed tightly on. In such a 100-day culture, population A, capable of growth in broth but not on plates, was estimated by broth dilution counts to be 7.60 logThese estimates suggest that the extent to which bacilli have gone into ill-defined dormancy might be measured as the proportion of a total bacterial population that can grow on plates. The lower this proportion, the greater the overall degree of "dormancy". Whatever, the theoretical significance of this simple technique for measuring dormancy, there is a practical implication for those undertaking long-term mouse experiments. Some end such an experiment with plate counts and others with culture in liquid medium. Those using only plate counts may be seriously underestimating the residual populations. It is also clear that there is much work to be done in seeing how various experimental conditions, such as the duration of the infection and the immune state of the mice, affect the ratio between broth and plate counts. Those exploring the development of new drugs need to know how these two populations respond to current anti-tuberculosis drugs and to novel drugs.The author(s) declare that they have no competing interests.All three authors took part in the running of the experiments with JD contributing the most. DAM contributed the concept of parallel broth and plate counts.The pre-publication history for this paper can be accessed here:


Soilborne wheat mosaic virus (SBWMV) 19K protein is a cysteine-rich protein (CRP) and shares sequence homology with CRPs derived from furo-, hordei-, peclu- and tobraviruses. Since the hordei- and pecluvirus CRPs were shown to be pathogenesis factors and/or suppressors of RNA silencing, experiments were conducted to determine if the SBWMV 19K CRP has similar activities. The SBWMV 19K CRP was introduced into the Potato virus X (PVX) viral vector and inoculated to tobacco plants. The SBWMV 19K CRP aggravated PVX-induced symptoms and restored green fluorescent protein (GFP) expression to GFP silenced tissues. These observations indicate that the SBWMV 19K CRP is a pathogenicity determinant and a suppressor of RNA silencing.Amino acid sequence analyses indicate that the  Cucumber mosaic virus (CMV) 2b, the Tobacco etch virus (TEV) HC-Pro, and the Tomato bushy stunt virus (TBSV) P19 [Viruses survive in their hosts either by evading or countering host defenses. Viral evasion is a passive mechanism by which viruses overwhelm host defenses, or invade organs or cells where the host defenses cannot reach them. The ability of a virus to counter host defenses requires an active mechanism to either bypass or disarm the host machinery. Viruses invading vertebrate hosts produce virokines and viroceptors which interact with immune response molecules to inhibit or modulate their anti-viral activities ,2. RecenBSV) P19 -10 proteBSV) P19 ,12.Furo-, Hordei-, Tobra-, Peclu-, Beny-, Carla-, and Pomovirus encode small cysteine-rich proteins (CRPs) near the 3' ends of their genomes, and some have been identified as both silencing suppressor proteins and pathogenicity factors. For example, the Barley stripe mosaic virus  gamma b protein and the Peanut clump virus  15K protein suppress RNA silencing, modulate symptom severity, and systemic virus accumulation [Tobacco rattle virus  16K CRP has been described as a pathogenicity factor and suppresses RNA silencing [Soilborne wheat mosaic virus  19K CRP, the BSMV gamma b protein, and the CMV 2b (which is not a CRP) protein functionally replaced the 16K CRP of TRV [Little is known about the evolution and phylogenetic relationships of silencing suppressor proteins. In particular, viruses belonging to the genera mulation -16. The ilencing . In compP of TRV .Furovirus [Tobacco mosaic virus (TMV) replicase [This study was undertaken to characterize the SBWMV 19K CRP. SBWMV is a bipartite RNA virus and is the type member for the genus eplicase . The 3' eplicase ,22. SBWMeplicase  and its eplicase . The CP eplicase . The 3' Potato virus X (PVX) infectious clone was used to express the SBWMV CRP and to study its role in virus pathogenicity and suppressing RNA silencing.To gain insight into the role of the SBWMV 19K CRP in virus infection, amino acid sequence comparisons were conducted to determine the relatedness of the SBWMV 19K CRP to other viral CRPs. The The Pfam Protein Families Database reports a family of CRPs with similar sequences which includes proteins from BSMV, PSLV, PCV and SBWMV Pfam 04521.5). Since there are viruses not included in the Pfam report that encode CRPs, this study was undertaken to determine if there is a larger CRP family containing related viral proteins. Further examination in this study reveals that the CRPs encoded by all known hordei-, peclu- and furoviruses share significant sequence similarity . The N-terminal halves of the aligned amino acid sequences, containing most of the Cys residues, have a mixture of extended, helical and loop predicted structures.The pecluviruses PCV and IPCV, and the hordeiviruses BSMV, LyRSV, and PSLV proteins contain a Ser-Lys-Leu sequence at the C-terminus. This tripeptide was shown for PCV to be a peroxisomal targeting signal . This siN. benthamiana, N. clevelandii, C. quinoa, and C. amaranticolor leaves .Immunoblot and northern analyses were conducted to verify PVX accumulation in the upper leaves of C. quinoa and C. amaranticolor leaves. Local lesions were detected in plants inoculated with PVX.GFP or PVX.19K between 5 and 7 dpi. PVX.19K-inoculated C. quinoa plants showed severe necrotic local lesions . RNA samples collected from non silenced and silenced plants were also tested by Northern analysis to confirm the systemic accumulation of PVX.GUS or PVX.19K (data not shown). Since, GFP expression was restored in plants systemically infected with PVX.19K but remained silenced in plants inoculated with PVX.GUS, it is likely that the SBWMV 19K ORF is a suppressor of RNA silencing.Northern analyses was conducted to confirm RNA silencing in the upper leaves of GUS Fig.  and 4J. Many viruses encode proteins that suppress RNA silencing but the phylogenetic relatedness of these proteins is poorly understood. In this study, one class of viral CRPs, which were described as suppressors of RNA silencing and/or viral pathogenicity determinants, were shown to be phylogenetically related. These CRPs have a conserved Cys-Gly-Xaa-Xaa-His motif in which one of the two Xaa residues is Lys or Arg. The N-terminus has several conserved Cys residues that likely comprise a zinc finger motif. In fact, the ability of the gamma b protein of BSMV to bind Zn(II) was recently demonstrated .Furovirus. As sequence data from different furoviruses have become available, it became clear that there are significant differences in the genome organization of these viruses, and therefore furovirus classification was revised in 1999 [Furovirus now consists of viruses similar in genome organization to SBWMV [Benyvirus, Pecluvirus, and Pomovirus, respectively, for two reasons [Furovirus and concluded that these small CRPs were unrelated [Benyvirus and inclusion of new members into the genus Furovirus led us to reexamine the relatedness of the viral CRPs. Based on the most recently defined taxonomic structure, the current amino acid sequence comparison presented in Figure Furo-, Hordei-, Peclu-, and Tobravirus are phylogenetically related. On the other hand, these proteins are so different from CRPs encoded by Pomo-, Beny- and Carlaviruses that the latter ones could not be included in the alignment  gamma b, TBSV P19, Tobacco etch virus HC-Pro, and the Rice yellow mottle virus P1 proteins[The present study shows that the SBWMV 19K CRP, when expressed from the PVX genome, functions as a pathogenesis factor and a suppressor of RNA silencing. The SBWMV 19K CRP, when it was expressed from the PVX genome, induced systemic necrosis on  proteins,11,14,34N. benthamiama, N. tabacum, C. quinoa, and C. amaranticolor (data not shown) plants, the SBWMV 19K CRP did not have any effect on symptomology (data not shown). However, it was reported previously that protein expression levels from the TBSV vector might be too low to test the effects of heterologous proteins on symptom severity [When we introduced the SBWMV 19K ORF into the TBSV vector and inoculated it to severity . Since aTobacco etch virus (TEV) and PVX in which the TEV HC-Pro protein enhances accumulation and disease severity of PVX [In a related study, the SBWMV 19K and the BSMV gamma b CRPs could substitute for the TRV 16K CRP within the TRV genome, promoting virus replication and systemic accumulation . The abiy of PVX . HC-Pro y of PVX .The phylogenetic relatedness of the hordei-, peclu-, and furovirus CRPs is further substantiated by evidence that these proteins are all capable of suppressing RNA silencing in emerging leaves. This was demonstrated in the present and related studies using the same reversal of silencing assay used in this study. The SBWMV 19K CRP, the BSMV and PSLV gamma b CRPs, and the PCV 15K CRPs were each unable to change GFP expression in leaves where GFP was silenced prior to virus infection. However in each case, GFP expression occurred in newly emerging leaves ,16. ThusChinese wheat mosaic virus (CWMV, a furovirus). A similar search began with the amino acid sequence of BSMV gamma b, a sequence recovered in the CWMV search. Both searches converged at the second iteration and retrieved the same set of 22 sequences. This set contained CRPs derived from furo-, peclu- and hordeiviruses and contained the conserved P18 PFAM domain  containing added sequences encoding a ClaI restriction site (underlined) and a reverse primer (CGC GTC GAC TCA CAA AGA GGA TAT CTT CTT TGG C) containing sequences encoding a SalI restriction site (underlined). PCR products and pPVX.GFP plasmids were digested with ClaI and SalI and then were ligated to prepare pPVX.19K.The SBWMV 19K CRP ORF was inserted into the PVX.GFP genome, replacing the GFP ORF. The 19K CRP ORF was reverse transcribed and PCR amplified from purified SBWMV RNA using a forward primer (GCG GGG In vitro transcription reactions contained: 0.25 μg of linearized DNA, 5 μl of 5X T7 transcription buffer, 1.0 μl of 0.1 M DTT, 0.5 μl of SUPERase·In™ ribonuclease inhibitor (20 U/ μl) (Ambion, Austin, TX), 2.5 μl of an NTP mixture containing 5 mM ATP, CTP, UTP, and GTP (Pharmacia-Pfizer, Mississauga, Ontario, Canada), 0.7 μl of T7 polymerase (Ambion), and nuclease-free water to a final volume of 25 μl. The reactions were incubated for one and a half hour at 37°C [ at 37°C .Nicotiana benthamiana, N. clevelandii, Chenopodium quinoa, and C. amaranticolor plants were inoculated with infectious transcripts to study disease severity. Four plants, two leaves per plant, were inoculated in each experiment. Experiments were repeated at least three times. Ten μl of undiluted PVX.GFP or PVX.19K transcripts were rub-inoculated to each plant.N. benthamiana line 16C was used to study RNA silencing. This line is homozygous for the GFP transgene at a single locus [Agrobacterium (see below).The transgenic le locus . Plants Agrobacterium tumefaciens strain C58C1 (pCH32) carrying a binary plasmid expressing GFP from a Cauliflower mosaic virus (CaMV) 35S promoter was used to silence GFP expression in N. benthamiana line 16C. Agrobacterium cultures were grown overnight at 28°C in 5 ml of L-broth medium containing 5 μg/ml of tetracycline and 50 μg/ml of kanamycin. This 5 ml culture was used to inoculate 50 ml L-broth and grown overnight in medium containing 5 μg/ml tetracycline, 50 μg/ml kanamycin, 10 mM MES, and 20 μM acetosyringone. Cultures of Agrobacterium containing GFP were pelleted by centrifugation and resuspended in a solution containing 10 mM MgCl2, 10 mM MES, and 150 μM acetosyringone. The final concentration of Agrobacterium was 0.5 OD600. The suspension was left at room temperature for 2–3 hours and then loaded into a 2 ml syringe. The syringe was used to infiltrate the suspension into the underside of the leaf.Agrobacterium and in PVX.GFP inoculated plants. GFP fluorescence was recorded with a Sony Digital Still Camera model DSC-F717 (Sony Corporation of America, New York City, New York). In all plants analyzed, GFP expression was monitored every 3 days for up to 21 days post inoculation (dpi) or post infiltration with Agrobacterium.A hand-held model B-100 BLAK-RAY long wave ultraviolet lamp (Ultraviolet Products, Upland, CA) was used to monitor GFP expression in 16C plants infiltrated with N. benthamiana leaves was extracted in 1:10 (w/v) grinding buffer (100 mM Tris-HCl pH 7.50, 10 mM KCl, 5 mM MgCl2, 400 mM sucrose, 10% glycerol, and 10 mM β-mercaptoethanol). Extracts were centrifuged at 10,000 g for 10 min. Equal volumes of supernatants and protein loading buffer (2 % SDS, 0.1 M dithiothreitol, 50 mM Tris-HCl pH 6.8, 0.1% bromophenol blue, and 10 % glycerol) were mixed and boiled for 5 min. SDS-PAGE was carried out for 1 h at 200 V using 30 μl of each sample and 12.5% SDS -PAGE and the Biorad Mini-Protean 3 system (Biorad Laboratories, Hercules, CA). Proteins were transferred to PVDF membranes (Amersham Biosciences Corp., Piscataway, NJ) at 4°C overnight using protein transfer buffer (39 mM glycine, 48 mM Tris base, 0.037% SDS, and 20% methanol, pH 8.3) and a BioRad Trans-Blot system (BioRad Laboratories). Immunoblot analyses were conducted using the ECL-Plus Western Blotting Detection Kit (Amersham Biosciences Corp.). PVX CP antiserum (1:200) (Agdia, Elkhart, IN) was used.Immunoblot analyses were conducted according to . Total pNorthern analyses were conducted according to . For anaNcoI and SalI and a 614 nt fragment was gel eluted and labeled using Dig High Prime kit (Roche Applied Science Inc. Indianapolis, IN). The CSPD DIG Luminescence Detection Kit (Roche Applied Science Inc.) was used for chemiluminescence detection of DIG-labeled probes. Special thanks to Wenping Qui at Southwest Missouri State University for assistance with studies using TBSV to express the SBWMV 19k.For detection of TBSV.GFP and TBSV.19K in infected plant extracts, a DNA probe was labeled with digoxigenin (DIG). TBSV.GFP plasmids were digested with BamHI and EcoRI and a 1 kb fragment corresponding to the 26S rRNA was recovered and labeled using the Dig High Prime DNA labeling system (Roche Applied Science Inc.).The p26SBE-2 plasmid was obtained from Kay Scheets at Oklahoma State University and contains the 26S ribosomal RNA gene in pBluescript. This plasmid was used to prepare a DNA probe for membrane detection of rRNA [45]. The p26SBE-2 plasmid was digested with The author(s) declare that they have no competing interests.Jeannie Te did all cloning, plant inoculation experiments, gene silencing experiments. Ulrich Melcher did the amino acid sequence alignments and phylogenetic comparisons. Amanda Howard did some gene silencing experiments, photography. Jeanmarie Verchot-Lubicz conceived the study, did some molecular analysis, and wrote the paper. Special thanks to Wenpiny Qiu at Southwest Missouri State University for assistance with studies using TBSV to express the SBWMV 19k.


Pituitary tumor transforming gene1 (PTTG1) is a novel oncogene that is expressed in most tumors. It encodes a protein that is primarily involved in the regulation of sister chromatid separation during cell division. The oncogenic potential of PTTG1 has been well characterized in the mouse, particularly mouse fibroblast (NIH3T3) cells, in which it induces cell proliferation, promotes tumor formation and angiogenesis. Human tumorigenesis is a complex and a multistep process often requiring concordant expression of a number of genes. Also due to differences between rodent and human cell biology it is difficult to extrapolate results from mouse models to humans. To determine if PTTG1 functions similarly as an oncogene in humans, we have characterized its effects on human embryonic kidney (HEK293) cells.We report that introduction of human PTTG1 into HEK293 cells through transfection with PTTG1 cDNA resulted in increased cell proliferation, anchorage-independent growth in soft agar, and formation of tumors after subcutaneous injection of nu/nu mice. Pathologic analysis revealed that these tumors were poorly differentiated. Both analysis of HEK293 cells transiently transfected with PTTG1 cDNA and analysis of tumors developed on injection of HEK293 cells that had been stably transfected with PTTG1 cDNA indicated significantly higher levels of secretion and expression of bFGF, VEGF and IL-8 compared to HEK293 cells transfected with pcDNA3.1 vector or uninvolved tissues collected from the mice. Mutation of the proline-rich motifs at the C-terminal of PTTG1 abolished its oncogenic properties. Mice injected with this mutated PTTG1 either did not form tumors or formed very small tumors. Taken together our results suggest that PTTG1 is a human oncogene that possesses the ability to promote tumorigenesis in human cells at least in part through the regulation of expression or secretion of bFGF, VEGF and IL-8.Our results demonstrate that PTTG1 is a potent human oncogene and has the ability to induce cellular transformation of human cells. Overexpression of PTTG1 in HEK293 cells leads to an increase in the secretion and expression of bFGF, VEGF and IL-8. Mutation of C-terminal proline-rich motifs abrogates the oncogenic function of PTTG1. To our knowledge, this is the first study demonstrating the importance of PTTG1 in human tumorigenesis. Pituitary tumor transforming gene 1 (PTTG1), a recently characterized oncogene, was initially identified on analysis of a rat pituitary tumor -4. ThreeTo date, the evidence for the oncogenic function of PTTG1 has been obtained by overexpression of PTTG1 in mouse fibroblast cells (NIH3T3) followed by assessment of its ability to induce cellular transformation in vitro (colony formation in soft agar) and tumor formation in nude mice ,4. Due tSimilarly, much of the evidence concerning the mechanisms by which PTTG1 may affect the phenotype of the cell has been obtained using transfected NIH3T3 cells. It is known that the secretion of growth factors and cytokines by tumor cells, and the cells that infiltrate and surround the tumor mass play an essential role in the regulation of tumor growth and metastasis . Both bFThe purposes of this study were, therefore, three-fold. Firstly, to determine whether PTTG1 can induce cellular transformation of normal human cells; secondly, to determine if PTTG1 is sufficient in itself to induce transformation; and, thirdly, to characterize the changes in secretion and expression of key metastatic, angiogenic and chemokine factors (bFGF, VEGF and IL-8) associated with PTTG1-mediated transformation in human cells. For these studies, we selected the human embryonic kidney HEK293) cell line as our model. The transformation of these cells by human adenovirus type 5 prevents their senescence . These c3 cell liHEK293 cells were transfected with pcDNA3.1-PTTG1, pcDNA3.1-mPTTG1 or pcDNA3.1 vector. After G418 selection, 10 clones from each of pcDNA3.1, pcDNA3.1-PTTG1 or pcDNA3.1-mPTTG1 transfected cells were picked, cultured and expanded. The PTTG1 protein expression of these transfectants was detected by western blot analysis using PTTG1 antiserum. Two representative clones from PTTG1 transfected (named HEKPTTG1-1 and HEKPTTG1-3) and mPTTG1 transfected (named HEKmPTTG1-2 and HEKmPTTG1-4), and one clone from pcDNA3.1 vector (named HEKpcDNA3.1) was selected for further studies. Selection of clones was based on the level of expression of PTTG1 protein. Fig. Previously, we have shown that over expression of PTTG1 in mouse fibroblast NIH3T3 cells results in an increase in cell proliferation . To deteAs anchorage-independent growth is considered to be an in-vitro test for angiogenesis we assayed the effects of transfection with PTTG1 on the ability of the HEK293 cells to form colonies in soft agar. As shown in Fig. 3 for HEKPTTG1-1, 72–1404 mm3 for HEKPTTG1-3 and 8.8–12.6 mm3 for HEKmPTTG1-2  followed by assessment of its ability to induce cellular transformation and tumor formation in nude mice ,4. HowevA second issue that we were able to address using the HEK293 cell model was the question of the ability of PTTG1 to induce transformation of normal human cells, i.e., whether it acts alone or in cooperation with another oncogene to achieve its tumorigenic function. It has been reported that a single oncogene may not be sufficient for induction of transformation but requires co-expression, or oncogenic cooperation, of another oncogene(s) to induce tumorigenesis in normal primary human cells ,25,42,43PTTG1 contains several-proline rich motifs (PXXP); two of these that are located in the C-terminal domain have been reported to be potential binding sites for SH3-domians . In our The molecular mechanisms by which PTTG1 achieves its tumorigenic function remain unclear. PTTG1 has been reported to induce expression of the c-myc oncogene , bFGF 1 and VEGFVEGF is a potent stimulant of the vascularization of tumors and is one of the most specific markers of tumor vasculature observed to date ,51. VEGFA direct correlation between high IL-8 expression and metastases in melanoma , ovarian163 to A163, P170 to A170 and P172 to A172, and P173 L173), was generated by site-directed mutagenesis using the Quick-change mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Mutation of these amino acids has been reported to abrogate the tumorigenic function of PTTG1 and to block the secretion and expression of bFGF in mouse NIH3T3 cells [The human embryonic kidney cell line HEK293), which had been transfected by exposing these cells to sheared fragments of adenovirus type 5 DNA  was purc93, which2 and 95% air. After incubation for 24 h, 48 h, 72 h or 96 h, 20 μl of dye solution from the kit was added to each well and incubated at 37°C for an additional 2 h. The quantity of formazon product was measured by its absorbance at 490 nm using a 96-well plate reader . Each experiment was repeated at least three times.Cell proliferation was assayed using the CellTiter 96 non-radioactive cell proliferation assay kit (Promega, Madison, WI) according to the manufacturer's instructions and as described previously . Briefly4) from each cell line were suspended in 0.3% agar in DMEM containing 10% fetal bovine serum and plated on solidified agar (0.7%) in 35 mm dishes. After 14 days of culture, colonies formed were counted and photographed as described previously [Anchorage-independent cell growth was determined by analyzing the formation of colonies in soft agar. Cells 10 from eac6/ml. The cells (1 × 106 cells in 200 μl PBS/site) were injected subcutaneously (s.c.) into both flanks of 5- to 6-week old female nu/nu mice (4 mice/group) (Charles River Laboratory, Wilmington, MA). All procedures were carried out following the protocol approved by The University of Louisville Institutional Animal Care and Use Committee. Four weeks after injection, the mice were scarified, and the tumors and other tissues harvested. The skin and connective tissues were dissected from the tumors, and the tumor volume was calculated from measurements of length × width × height. The tissues were divided into two parts, one part being fixed in 10% buffered formalin and the other stored in liquid nitrogen. For histopathologic analysis, 5 μm sections were cut from paraffin-embedded tissues, and mounted on slides. Sections were stained with H&E [Cells growing in log phase were harvested by trypsinization and washed twice with PBS. The cells were resuspended in PBS to a final concentration of 5 × 10with H&E , and pro3VO4, and 1 mM NaF) supplemented with Complete Mini Protease Inhibitor tablets . Equal amounts of protein extracts (40 μg) were resolved on 12% SDS-PAGE gel, and transferred onto a nitrocellulose membrane (Amersham, Piscataway, NJ). Blots were probed with PTTG1 antiserum at a dilution of 1:1,500 as described previously [Cells growing in log phase were lysed in chilled lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 1 mM Naeviously . ImmunorThe levels of bFGF, VEGF and IL-8 in tissue culture supernatants and tissue homogenates were measured using commercially available ELISA kits from BD Biosciences (Minneapolis, MN). To measure bFGF, VEGF and IL-8 in the culture supernatants, HEK293 cells were transiently transfected with pcDNA3.1 or pcDNA3.1-PTTG1 cDNA using Fugene6 as the transfectant reagent as described previously . After 2Total RNA from tumors and other tissues was purified using Trizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. The RNA pellets were resuspended in RNase-free water, and the contaminating DNA was removed from the preparations with DNaseI. The yield of total RNA was measured using a spectrophotometer, and the quality was assessed by electrophoresis through a 1% agarose gel. First strand cDNA was synthesized using the iScript™ cDNA synthesis kit (BioRad, Hercules, CA). PCR primers Table  were desIn summary, our results demonstrate that PTTG1 is a potent human oncogene and has the ability to induce cellular transformation of human cells. Overexpression of PTTG1 in HEK293 cells leads to an increase in the secretion and expression of bFGF, VEGF and IL-8. Mutation of C-terminal proline-rich motifs abrogates the oncogenic function of PTTG1. To our knowledge, this is the first study demonstrating the importance of PTTG1 in human tumorigenesis.TH carried out the in-vivo studies, RT-PCR analysis, western blot analysis and drafted the manuscript. MTM generated stable clones for HEKmPTTG1-2 and 4, RT-PCR and ELISA analysis. SSK participated in study design and coordination and generated stable cell lines for HEKPTTG1-1 and 3. All authors read and approved the final manuscript.


Here, we isolated tumor-sensitized T cells and activated them in vitro using conditions that led to greater than 108-fold numerical hyperexpansion of either the CD4+ or CD8+ subset while retaining their capacity for in vivo therapeutic efficacy. Murine tumor-draining lymph node (TDLN) cells were segregated to purify the CD62Llow subset, or the CD4+ subset thereof. Cells were then propagated through multiple cycles of anti-CD3 activation with IL-2 + IL-7 for the CD8+ subset, or IL-7 + IL-23 for the CD4+ subset. A broad repertoire of TCR Vβ families was maintained throughout hyperexpansion, which was similar to the starting population. Adoptive transfer of hyper-expanded CD8+ T cells eliminated established pulmonary metastases, in an immunologically specific fashion without the requirement for adjunct IL-2. Hyper-expanded CD4+ T cells cured established tumors in intracranial or subcutaneous sites that were not susceptible to CD8+ T cells alone. Because accessibility and antigen presentation within metastases varies according to anatomic site, maintenance of a broad repertoire of both CD4+ and CD8+ T effector cells will augment the overall systemic efficacy of adoptive immunotherapy.T cell-mediated cancer immunotherapy is dose dependent and optimally requires participation of antigen-specific CD4 Cancer immunotherapy, using T lymphocytes that recognize tumor-specific antigens, holds great promise. Advantageous features include: exquisite specificity for targeted antigens, thereby sparing normal tissues, and the ability of effector T cells to traffic to tumor in all anatomic locations. Although most effector T cells are subject to activation-induced cell death (AICD), a memory response is established leading to sustained protection . DespiteCognizant of these features, many preclinical studies of active immunotherapy have used a vaccination/challenge scheme to avoid tumor-induced immunosuppression or have alternatively treated hosts with minimal tumor burdens. Several human clinical trials have similarly focused on hosts with minimal residual disease in order to define the magnitude and characteristics of the immune response. These studies have clearly established that immune responses are successfully generated in vaccinated cancer patients. However, the frequency of responding T cells is typically less than one percent even after multiple cycles of vaccination -14. In cex vivo activation of purified CD62Llow T cells from TDLNs generates potent effector CD4+ and CD8+ T cells that can mediate regression of advanced tumors in every tested anatomic location [+ versus CD8+ effector cells also varies considerably between pulmonary metastases and intracranial (i.c.) or subcutaneous (s.c.) tumors [+ as well as CD8+ tumor-reactive effector T cells would be required for optimal adoptive immunotherapy against disseminated metastatic disease.Adoptive immunotherapy is another approach to cancer immunotherapy that circumvents some of the limitations of active immunotherapy. Animal tumor models have convincingly demonstrated that hosts bearing progressively growing weakly immunogenic tumors nevertheless generate sensitized T cells in TDLN . Antigenlocation -26. The ) tumors . This inin vitro activation of tumor-sensitized L-selectinlow precursors with anti-CD3 mAb and high concentrations of IL-2 (100 U/ml) induced rapid proliferation of CD8+ effector cells [3-fold. In this report, we define ex vivo activation conditions that permit numerical expansion of either CD4+ or CD8+ effector T cells to greater than 108-fold while retaining their high therapeutic potency and preserving a broad T cell receptor (TCR) repertoire.We investigated whether we could overcome quantitative limitations associated with active immunotherapy through extensive numerical expansion of effector cells. In a previous study, we determined that or cells . AdoptivFemale C57BL6N (B6) mice were purchased from the biologic Testing Branch, National Cancer Institute (Frederick, MD). They were maintained in a specific pathogen-free environment according to National Institute of Health guidelines. Mice were used for experiments at 8–10 weeks of age. The MCA 205 and MCA 207 fibrosarcomas, syngeneic to B6 mice were serially passaged in vivo s.c. as described previously .6 MCA 205 cells and 12 days later the TDLNs were removed and mechanically disrupted to obtain a single cell suspension. The TDLN cells were incubated with 100 μl anti-CD62L microbeads per 108 cells and applied to MACS columns (Miltenyi Biotech, Auburn CA) and the flow through fraction was collected. For CD4+ hyperexpansion, the CD62Llow subset was depleted of CD8+ cells by MACS on day 0 and day 36 of culture activation. CD62Llow cells, containing approximately 50% TCR+ and 50% B220+ subsets, were suspended in complete medium (CM) and incubated for 2 days at 4 × 106 per well in 24 well culture plates coated with anti-CD3 (145-2C11) as previously described [5/ml in CM with IL-2 (4 U/ml) (Chiron Corp. Emeryville, CA), with or without rmIL-7 (10 ng/ml) or rhIL-23 (2 ng/ml) (each from R&D Systems, Minneapolis, MN) and then diluted to 105/ml on day 5 of activation. On days 9 and 15, the cell concentration was adjusted to 2 × 105/ml. For experiments with two cycles of anti-CD3 stimulation, T cells were incubated with immobilized anti-CD3 for 14 hrs on day 15 and used for adoptive therapy on day 23. For long-term expansion, cultures were maintained for 23 days after the initial anti-CD3 stimulation in CM with the indicated combination of IL-2 (4 U/ml), IL-7 (10 ng/ml), and IL-23 (2 ng/ml) and then were stimulated with anti-CD3 for 14 hrs on day 23 and every 7 days thereafter.Tumors were established by s.c. flank inoculation of 1.5 × 10escribed . ActivatT cells were stimulated with a single cell suspension of either MCA 205 or MCA 207 tumors at a 1:1 ratio, or with immobilized anti-CD3. Brefeldin A was added after five hours of stimulation and the cells were harvested after 20 hrs and stained for intracellular IFN-γ according to the manufacturers instructions (BD Biosciences, San Diego, CA). FACS analysis was performed using FITC or PE conjugated antibodies or isotype control antibodies (BD Biosciences).TDLN cells were lysed using TRIzol reagent (Invitrogen, Carlsbad, CA) and total RNA was reverse transcribed into cDNA using the SuperScript II RT kit (Invitrogen). cDNA was amplified using PCR with 22 different VB-specific primers paired with a hex-labeled constant region primer which spans the CDR3 region as previously described . CDR3 si5) i.v. to establish pulmonary metastases. Subcutaneous tumors were established by inoculation of 1.5 × 106 cells. Intracranial tumors were established by transcranial inoculation of 105 tumor cells at a depth of 4 mm as previously described [137Cs irradiator prior to intravenous transfer of the T cells whereas mice with 3-day pulmonary tumors were not irradiated. For pulmonary tumors, mice were euthanized on day 20 post inoculation, the lungs were insufflated with India ink and the number of surface tumor nodules was enumerated using a dissecting microscope. Subcutaneous tumors were measured in two perpendicular dimensions three times per week and mice with progressive tumors were euthanized when the product of dimensions exceeded 200 mm2. Mice bearing intracranial tumors were monitored daily for survival or were euthanized when neurologic symptoms such as decreased grooming and decreased spontaneous movement were apparent.Mice were inoculated with MCA 205 or MCA 207 tumor cells  with or without IL-7 (10 ng/ml) and the cell concentration was adjusted to 105/ml on day 5 and 2 × 105/ml on day 9. As demonstrated in figure low B220+ cells. This was followed by a rapid burst of proliferation from day 2 until day 15 when the IL-2 supplemented culture peaked at 175-fold proliferation and the IL-2 + IL-7 cultures reached 1000-fold proliferation.During the progressive growth of weakly immunogenic tumors an immune response, albeit sub-therapeutic, is initiated in TDLNs. In previous studies our laboratory has demonstrated that the CD62Lactivity ,24,32. Tactivity -23. TDLN6 CD62Llow TDLN cells is near the lowest threshold dose required to cure 3-day s.c. MCA 205 tumors [in vitro activation augmented the total number of cells but did not substantially diminish per-cell therapeutic efficacy.The morphology of the cells changed from lymphoblastoid to small round cells at day 15 and there was no additional proliferation. IL-7 preserved the viability of cells whereas IL-2 alone could not prevent a 8-fold numerical decline between days 15 to 30. Because the TDLN cells were initially segregated based on phenotype rather than antigen specificity and the anti-CD3 stimulation was antigen-independent, it was not known whether the enhanced proliferation achieved in the presence of IL-7 was due to preferential growth of irrelevant T cells or preservation of tumor-reactive T cells. As demonstrated in figure 5 tumors ,33. Thus+ T cells [+ and CD8+ T cells each proliferated in the presence of IL-2 and IL-7, by day 23 CD8+ cells comprised 86% of the culture whereas there were only 4% CD4+ cells  "see 8-fold total proliferation had occurred. In addition, TCR spectratype analysis for each Vβ family revealed a poyclonal rather than clonal or oligoclonal distribution (data not shown).As demonstrated in Figure + and CD8+ indicating preferential expansion or survival of CD8+ cells under the conditions employed. As shown in Figure 7 cells to hosts with 3-day MCA205 pulmonary metastases eliminated tumors and 6 × 106 cells was the threshold dose for complete response whereas 2 × 106 cells were subtherapeutic. In addition, there was no response against the antigenically distinct MCA207 tumor. In an independent experiment, the dose of T cells required to completely eliminate 3-day pulmonary metastases was 2 × 106 indicating some inter-experimental variability in per-cell efficacy. Because of the critical role of CD4+ T cells for therapy of s.c. or i.c. tumors it was not anticipated that the hyperexpanded CD8+ cultures would mediate complete regression of tumors at these anatomic sites. Indeed, there was substantially less efficacy against 3-day s.c.tumors. Adoptive transfer of 4 × 107 cells showed a trend toward response (P = 0.061) with 1/5 mice cured in only one of two identically designed experiments   with 1/5 mice achieving complete tumor regression.The CD4) Figure . As previn vivo was driven by tumor-specific antigens but the segregation of the CD62Llow subset and all subsequent in vitro activation stimuli were antigen independent. Consequently, reactivity of cultures against tumor antigens might fluctuate over time. This possibility was analyzed by quantifying the percentage of culture activated T cells that produce IFN-γ specifically when exposed to tumor in vitro. For this assay, a single cell digest of in vivo propagated tumor is used which contains MHC class II+ APC as well as tumor cells and is capable of stimulating both CD4+ and CD8+ T cells. As demonstrated in Figure + T cells produced IFN-γ in response to MCA 205 tumor cells. This percentage of IFN-γ positive CD8+ cells decreased to 10% on day 36. CD8+ T cells that were simply maintained in culture with IL-2 and IL-7 cytokine support but without anti-CD3 restimulation did not proliferate but remained viable. Under non-proliferative conditions, the percentage of MCA 205 reactive T cells was maintained at 13% on day 36. Similarly, CD4+ T cells cultured in the presence of IL-7 plus IL-23 had 11% IFN-γ positive cells in response to MCA205 tumor with minimal spontaneous production and minimal response to MCA207 tumor digest. The in vitro assay of IFN-γ production may not be fully reflective of the in vivo capacity of T cells to mediate tumor regression because CD4+ T cells cultured with IL-2 plus IL-7 had only 2% IFN-γ positive cells despite equivalent in vivo anti-tumor efficacy. The vast majority of T cells (73–86%) produced IFN-γ in response to anti-CD3 stimulation throughout the in vitro culture activation period.The initial antigen priming event in vivo, can be activated in vitro under conditions that promote hyperexpansion of either the CD4+ or CD8+ subset while retaining their potent therapeutic efficacy against established tumors. A notable feature of in vitro activation is that it permits selection and enrichment of a minor subset of tumor-reactive precursor cells. The mechanism of antigen sensitization of T cells through cross-priming by APC within draining LNs provides a convenient localized anatomic source that is already highly enriched. When coupled with physical segregation based on phenotypic characteristics that distinguish between antigen-stimulated versus naïve T cells, enrichment to nearly 40% of tumor-specific T cells was achieved. One important aspect of this strategy for selection and enrichment is that it does not require pre-existing knowledge of the immunogenic tumor antigens and does not require freshly acquired T cells to exhibit effector function. These conditions have relevance for many clinical situations where tumor antigens are not yet described or where unique tumor antigens may be immunodominant. Moreover, signaling defects have been observed in freshly acquired T cells from tumor-bearing hosts that might impede segregation based on functional properties [These experiments demonstrate that T cells, sensitized to tumor antigens operties -44.in vivo. The principal advantage to in vitro activation is that the culture conditions can be adapted to optimize proliferation of distinct subsets of responding cells. It is important to note that anti-CD3 activation in the absence of exogenous cytokine support did not lead to T cell proliferation even among CD4+ cells. The low cell density may have prevented secreted cytokines from reaching a critical threshold concentration. Moreover, IL-7, produced by non-hematopoetic cells and IL-23, produced by APC, mandated an exogenous source of these cytokines for in vitro culture activation. The combination of IL-2 and IL-7 provided rapid proliferation of CD8+ T cells and preserved their viability after completion of the initial mitogenic burst. The reason this combination was effective is that IL-7 receptor α chain is constitutively expressed on naïve and memory T cells but is downregulated on activated T cells [in vivo cytokine administration. More importantly, we have clearly documented that adjunctive IL-2 inhibits trafficking of adoptively transferred T cells into intracranial or subcutaneous tumors [in vitro without adverse systemic effects on the host. Future experiments to adjust the sequence, and concentration of supplemental cytokines using more sophisticated schedules than employed here might provide superior effector function.Starting with a highly enriched population of T cells we were able to use a powerful, yet antigen-independent, stimulus such as anti-CD3 mAb that preserved the initial TCR repertoire diversity. Interestingly, anti-CD28 stimulation was not required for this experimental model, presumably because the T cells had already received co-stimulation during APC-mediated priming  T cells ,46. By c T cells . Likewis T cells . Howevers tumors . By cont+ T cells for therapy of tumors in certain anatomic sites, such as the brain and subcutaneous tissue. The slower rate of CD4+ cell proliferation relative to CD8+ cells following the initial anti-CD3 stimulation led to their rapid marginalization in mixed cultures. However, depletion of CD8+ cells and use of cytokine combinations such as IL-7 and IL-23 favored the selective hyperexpansion of CD4+ cells that retained potent in vivo function. IL-23 is a member of the IL-12 family of cytokines and contains the IL-12 p40 subunit that transduces signals through the shared IL-12β1 chain in addition to the unique p19 subunit [+ cells, thus it is ideal for previously sensitized LN T cells. Myeloid cells, which are the natural source of IL-23, disappear rapidly in the in vitro cultures mandating an exogenous source. There is not a substantial amount of data on the effects of in vivo IL-23 administration, therefore its utility as a systemically administered adjuvant for T cell adoptive immunotherapy is unclear. However, the related cytokine IL-12 has substantial systemic effects that have limited its clinical use [These experiments confirm the importance of CD4 subunit ,51. The ical use .+ T cells have been investigated as a source of helper function for CD8+ cytolytic cells our previous experiments have clearly established their stand-alone potential against MHC class II negative tumors [+ T cell anti-tumor function is mediated through cross-presentation of specific tumor-antigens by tumor associated APC [+ cells cultured with IL-23 produced greater levels of INF-γ that would augment antigen presentation. Indeed, addition of tumor-reactive CD4+ cells to tumor digest increases the reactivity of the CD8+ cells. In addition to their autonomous effector functions, CD4+ cells are required to generate a functional CD8+ memory response in vivo [+ cells might be particularly useful because it, unlike IL-12, induces production of the pro-inflammatory cytokine IL-17 [+ T cells and inflammatory conditions of tumor antigen acquisition by host APC are important in perpetuating the anti-tumor response.Although CD4e tumors . CD4+ T ated APC . As demo in vivo ,56. Our  in vivo . In thisne IL-17 . Our obsin vitro activation first under conditions that optimize T cell proliferation, then with conditions that restore effector functions immediately before adoptive transfer, would be advantageous. Future experiments will explore whether it is possible to dissociate proliferative signaling pathways from those mediating effector function through selective transient gene inactivation. The quantitative aspects of hyperexpansion are of less scientific interest but do have some practical implications. The 108-fold extent of proliferation far exceeded what was required to treat the tumor models employed. Moreover, the availability of uniformly primed T cells for mechanistic studies is not numerically limited when using an inbred strain. However these experiments establish an approach to maintain polyclonality and preserve effector function despite extensive antigen-independent proliferation. As such, the quantitative aspects of hyperexpansion may have relevance to certain clinical situations where an autologous source of antigen-primed T cells may be limited and extensive host tumor burden may demand a large number of effector T cells.Repetitive anti-CD3 stimulation was utilized to drive hyperproliferation of T cells yet the TCR/CD3 complex also activates genetic programs required for effector function. Effector molecules such as Fas, Perforin, and IFN-γ have autoinhibitory as well as paracrine inhibitory effects during culture activation. Viewed purely in operational terms, sequential This is a table describing the percentage of cells expressing various TCR Vβ family members at three different time points.Click here for file