Individuals with chronic fatigue syndrome (CFS) have been shown to have reduced activity levels associated with heightened feelings of fatigue. Previous research has demonstrated that exercise training has beneficial effects on fatigue-related symptoms in individuals with CFS.The aim of this study was to sustain an increase in daily physical activity in CFS patients for 4 weeks and assess the effects on fatigue, muscle pain and overall mood.Six CFS and seven sedentary controls were studied. Daily activity was assessed by a CSA accelerometer. Following a two week baseline period, CFS subjects were asked to increase their daily physical activity by 30% over baseline by walking a prescribed amount each day for a period of four weeks. Fatigue, muscle pain and overall mood were reported daily using a 0 to 100 visual analog scale and weekly using the Profile of Mood States (Bipolar) questionnaire.3 counts/day vs. 267.2 ± 79.5 × 103 counts/day) during a 2-week baseline period. At baseline, the CFS patients reported significantly (P < 0.01) higher fatigue and muscle pain intensity compared to controls but the groups did not differ in overall mood. CFS subjects increased their daily activity by 28 ± 19.7% over a 4 week period. Overall mood and muscle pain worsened in the CFS patients with increased activity.CFS patients had significantly lower daily activity counts than controls (162.5 ± 51.7 × 10CFS patients were able to increase their daily physical activity for a period of four weeks. In contrast to previous studies fatigue, muscle pain, and overall mood did not improve with increased activity. Increased activity was not presented as a treatment which may account for the differential findings between this and previous studies. The results suggest that a daily "activity limit" may exist in this population. Future studies on the impact of physical activity on the symptoms of CFS patients are needed. Chronic fatigue syndrome (CFS) is characterized by persistent debilitating fatigue often accompanied by a complex of other symptoms  lasting at least six months that is unresolved with rest or medication ,2. A priIt is widely accepted that a sedentary lifestyle may greatly increase the risk of development of cardiovascular disease and Type II diabetes, as well as contributing to bone loss, and an age-related loss of function in a person's ability to perform daily activities -9. RecenFew randomized controlled trials designed to assess the efficacy of exercise training have been conducted in the CFS population. Two studies used graded exercise performed several days-a-week over a period of 12–26 weeks and both reported an improvement in fatigue-related symptoms and aerobic capacity ,12. AddiThe purpose of this study was to assess normal daily physical activity levels in CFS patients using accelerometers over a two-week period and compare these values to a group of healthy sedentary individuals. Additionally, the CFS patients were asked to increase their daily physical activity approximately 30% over a four week period by walking a prescribed amount each day while maintaining their non-exercise daily physical activity. Daily ratings of mood, fatigue, and muscle pain intensity were assessed and compared between the groups as well as in response to increased daily activity in the CFS patients.All experimental procedures were approved by the Institutional Review Board at the University of Georgia, and informed consent was obtained from each participant. All participants were recruited from the general community and either responded to a newspaper ad, responded to a flyer placed around campus, or were referred to the study by their physician. Seventeen CFS and twenty-one controls responded and were screened as possible study participants. A physician's diagnosis of CFS was required for inclusion. Additionally, CFS patients were required to confirm a self-report of decreased physical activity compared to pre-CFS levels, and self-reported inability to sustain high levels of physical activity without a subsequent exacerbation of CFS symptoms for study inclusion. CFS participants with a self-reported history of depression or other psychiatric illness were excluded. Control participants were chosen to be similar in age, height, and weight to the CFS patients and were defined as sedentary by self-report of one bout of regular exercise per week or less. The most sedentary participants (those with desk jobs, etc.) were given first priority for study inclusion. Control participants were also apparently healthy and reported no illnesses or disease conditions. Medications were monitored in all participants. The CFS participants were found to be taking many medications, both prescription and over-the-counter. Analgesics such as Vioxx, Celebrex, Advil, and Aleve were common.Initially, participants received instructions for wearing the activity monitors and for completing a daily activity log. A "pre" score from the 30-item Profile of Mood States short form questionnaire (POMS) was obtained . Participants proceeded to wear the monitors for two weeks during which time they were instructed to maintain normal daily activity. After the two weeks, data were collected from the activity monitors, and the monitors were reset. The participants then wore the monitors for an additional four weeks. The CFS patients were asked to increase their daily physical activity (30% above baseline) during this four week period by walking a prescribed amount each day in order to approximate the daily physical activity of a healthy sedentary person. This was based upon averaging the findings of others that suggested CFS patients had activity levels that were 15% to 40% reduced from healthy but sedentary individuals . CFS patDaily physical activity was assessed by a CSA accelerometer . To ensure accurate measurements, the procedure recommended by CSA was followed – the monitors were positioned over the subject's anterior superior iliac spine with the belt fitting snuggly so as to limit extraneous monitor movement. Participants were asked to wear the monitors at all times of the day, including sleep. Two minute epochs were used. Data were retrieved from the monitors using a specially designed docking module that input data into a computer.Recommended percent increases in daily activity were calculated based on each subject's average daily counts during their two-week baseline activity period. Counts are arbitrary units assigned to movements detected by the accelerometer. Counts are assigned based upon the magnitude of a change in velocity during a given time period. The number of counts needed to raise daily activity approximately 30% was calculated. Participants were then asked to walk on a treadmill at what they considered to be a comfortable walking pace. Walking speed was recorded, and used to calculate the recommended daily walking time. The approximate number of counts per minute for various walking speeds was assessed prior to the onset of the study (unpublished observations). Additionally, a pedometer was also used to aid participants in achieving the desired daily activity increase. Participants were given an approximate number of steps to take each day during their walk based upon their prescribed walking pace and time. Steps per minute for various walking speeds were assessed in a similar manner as counts per minute prior to the study (unpublished observations).Data concerning daily activity, mood, fatigue, and muscle pain were obtained from each subject via daily self-report. Participants were asked to report all daily activities and time spent engaged in each. Time periods when the activity monitor was not worn were also reported. A series of five questions concerning daily mood, fatigue intensity, and muscle pain intensity were also answered. Participants ranked, using a 10 cm (0 to 100 mm) visual analog scale their general overall daily mood for that day , the intensity of their fatigue that day (with 0 being no fatigue and 100 being the highest intensity fatigue imaginable), and the intensity of their muscle pain that day (with 0 being no pain and 100 being the worst imaginable pain). A similar visual analog scale has been used previously in CFS patients to rate daily fatigue . AdditioIndependent samples t-tests were conducted to compare for differences in subject characteristics between the groups. A repeated measures ANOVA was conducted to determine differences between activity level and mood in CFS and control participants. When a significant interaction was observed a one-way repeated measure ANOVA was performed to analyze simple effects with planned comparisons performed to analyze differences in treatment means. All values are reported means ± SD. Analyses were conducted and significance was assumed at an α level of 0.05.No participants (CFS or controls) reported adverse events associated with the increased activity program. Data were obtained from six CFS patients as well as seven sedentary control participants. Additionally, two other CFS patients began the testing protocol but were removed from the study at their own request. One was removed on doctor's recommendation due to an injury (unrelated to the study) and the second was removed due to a change in residence. The physical characteristics of all participants are presented in Table P = 0.017). All six of the CFS participants were successful in increasing their daily physical activity. Their daily activity counts increased 28%, on average, during the four-week training period (P < 0.001). However, it should be noted that 4 of the 6 CFS participants did not reach the prescribed 30% increase in daily activity. Interestingly, following their activity increase the CFS participants had activity levels that were still 24% reduced from those of the control group (P = 0.08).Individual as well as mean group daily activity counts are presented in Table Figures P = 0.016, Eta2 = 0.311) in overall mood. CFS participants reported a worsening of overall mood over time compared to controls. Neither an interaction nor a time main effect was observed in ratings of fatigue intensity. A significant group difference was observed between the CFS and control participants . Although not statistically significant, ratings of fatigue intensity in the CFS group did increase from 58.2 ± 8.5 to 67.0 ± 17.5 (indicating a worsening of symptoms). A significant group-by-time interaction (P = 0.03, Eta2 = 0.295) was seen between the CFS and control participants in their ratings of muscle pain intensity . Additionally, the CFS participants also reported experiencing a significantly greater amount of time spent with muscle pain each day . A significant time main effect was found for time spent with fatigue each day (P = 0.047, Eta2 = 0.243). A significant difference was found between baseline activity, 451 ± 528 min/day, compared to the final two weeks of increased activity 521 ± 566 min/day (P = 0.048, Eta2 = 0.287). The CFS participants also demonstrated a non-significant increase in time spent with muscle pain each day during baseline activity, the first two weeks of increased activity and the final two weeks of increased activity (554 ± 507 min/day vs. 642 ± 546 min/day vs. 713 ± 557 min/day). Control participants demonstrated no change over time in time spent each day with fatigue or muscle pain.The amount of time spent with fatigue each day as well as the amount of time spent with muscle pain each day was also reported by both groups. During baseline activity, the CFS participants reported experiencing a significantly greater amount of time spent with fatigue per day compared to the control participants . No change was observed in the fatigue scores of the CFS participants as their daily physical activity was increased. The control participants also demonstrated no change over time. Similarly, a significant difference was also observed between the CFS and control groups when vigor scores were compared (P = 0.036, Eta2 = 0.343). No change was observed in the vigor scores over time or in response to increased activity in either group.Figures 4A primary finding of this study was that individuals with CFS can increase their daily physical activity 28% on average over a four week period. Average daily activity counts, measured by accelerometer, increased in all six of the CFS participants who participated in the study. The magnitude of the observed increases in daily activity ranged from approximately 13% to 60%. These results are consistent with previous studies that have shown that CFS patients can sustain training programs lasting 12 to 26 weeks ,12. The Another interesting aspect of this study was the fact that even after increasing their daily activity 28%, our CFS participants were still approximately 25% less active than our sedentary control participants. We attempted a 30% increase in daily physical activity with the hope of bringing the daily activity levels of our CFS participants up to those of sedentary individuals. This was based upon the findings of others that suggested CFS patients had activity levels that were 15% to 40% reduced from healthy but sedentary individuals . Our resTo our knowledge, this study was one of the first to obtain daily ratings of fatigue intensity from a group of CFS participants under normal daily physical activity conditions as well as during periods of increased daily physical activity. Consistent with their diagnosis, CFS patients reported much greater daily ratings of fatigue intensity, time spent each day with fatigue, as well as fatigue recalled during the prior week compared to healthy sedentary participants. This large difference was expected based on the diagnosis and demonstrated the usefulness of ratings of this type to confirm fatigue symptoms.We found that overall mood, muscle pain intensity, and time spent each day with fatigue worsened following increased activity in our CFS participants compared to controls. Additionally, daily ratings of fatigue intensity (VAS) increased moderately over time while weekly fatigue (POMS) remained stable but elevated in the CFS patients. These observations are contrary to previous studies of increased exercise in this population where exercise training has been shown to reduce fatigue-related symptoms ,12. ThesIt is not clear why our study did not find increased daily physical activity to reduce symptom severity, as reported in previous studies -13. One Presentation of exercise as a possible treatment for CFS symptoms could have played a role in the improvements in symptom severity observed with exercise training -13. CognA third explanation of our findings is that there may have been something inherent in our exercise protocol that prevented fatigue symptoms from improving. Our method of activity increase, self-paced walking, was no more strenuous than the exercise used in other studies -13. HoweOur prescribed exercise was less intense and occurred over a shorter time period than that of previous studies -13. It iFive of our six CFS participants also reported having fibromyalgia. Fibromyalgia is a related syndrome with a primary symptom of muscle pain and tenderness . Interes2 values from 0.101 to 0.377), suggesting that more participants would be needed to detect differences between the subject groups. Consequently, additional studies will be needed to confirm our results.It is important to note that our small sample size is a key limitation in our study. The study is under powered to detect differences between our groups. Most of the measured variables had small to moderate effect sizes (EtaIn conclusion, this study found that individuals with CFS were able to increase their daily physical activity by approximately 28% for four weeks without serious health complications. At baseline activity our CFS participants exhibited significantly lower daily activity than sedentary controls. This reduced level of daily activity was larger than previously reported values,4,6. Lar


Traditional methods of measuring oral health mainly use clinical dental indices and have been complemented by oral health related quality of life (OHRQoL) measures. Most OHRQoL studies have been on adults and elderly populations. There are no systematic OHRQoL studies of a population-based sample of children. The objective of this study was to assess the prevalence, characteristics and severity of oral impacts in primary school children.Cross-sectional study of all 1126 children aged 11–12 years in a municipal area of Suphanburi province, Thailand. An OHRQoL measure, Child-Oral Impacts on Daily Performances index (Child-OIDP) was used to assess oral impacts. Children were also clinically examined and completed a self-administered questionnaire about demographic information and oral behaviours.89.8% of children had one or more oral impacts. The median impact score was 7.6 and mean score was 8.8. Nearly half (47.0%) of the children with impacts had impacts at very little or little levels of intensity. Most (84.8%) of those with impacts had 1–4 daily performances affected (out of 8 performances). Eating was the most common performance affected (72.9%). The severity of impacts was high for eating and smiling and low for study and social contact performances. The main clinical causes of impacts were sensitive tooth (27.9%), oral ulcers (25.8%), toothache (25.1%) and an exfoliating primary tooth (23.4%).The study reveals that oral health impacts on quality of life in Thai primary school children. Oral impacts were prevalent, but not severe. The impacts mainly related to difficulty eating and smiling. Toothache, oral ulcers and natural processes contributed largely to the incidence of oral impacts. Contemporary concepts of health suggest that dental health should be defined in physical, psychological and social well-being terms in relation to dental status ,2. That A number of socio-dental or OHRQoL measures have been developed and used for assessing oral well-being and to describe oral impacts on people's quality of life . GeneralMost studies using OHRQoL to assess oral impacts of the mouth and teeth have been on adults and elderly populations. Few studies have been done on children possibly because no OHRQoL measures designed for use with children existed until recently. A single measure, dental pain, has been used on children in Malaysia  and in SA cross-sectional survey was carried out in a municipal area of Muang district, Suphanburi province, Thailand. The sample was all 1,126 students aged 11–12 years, in the final year class of all primary schools (grade 6) in the area.Data were collected through: a) an interview for oral impacts using the Child-OIDP , by one The protocol of the study was approved by the Ethical Committee of the Ministry of Public Health of Thailand. Primary education and local health authorities as well as all primary schools in the study areas gave permission. Positive consent forms and letters informing parents were sent to parents.Two comprehensive OHRQoL measures specifically for use with pre-adolescent children have recently been developed; the Child Perceptions Questionnaire (CPQ11-14)  and the The procedure for using the Child-OIDP began with a self-administered questionnaire carried out with all children as a group in their classroom. The questionnaire contains a list of all oral problems that children are likely to perceive and also include an open answer for any unexpected perceived problem. It was developed during a pilot study, as a modification from the one used in the original OIDP. Children were asked to identify oral problems that they perceived in the last three months. This step aimed to focus children's attention to their oral health problems and to lead to the oral impacts assessment later. Their answers here were used only as a guide to investigate oral impacts on daily performances in the next step and were referred to when they were asked about the causes of oral impacts in individual interviews. Thereafter, children were individually interviewed, irrespective of their answers at the first step, to assess oral impacts on daily life in relation to 8 daily performances. The 8 performances were: a) eating, b) speaking, c) cleaning teeth, d) relaxing, including sleeping, e) smiling, laughing and showing teeth without embarrassment, f) maintaining emotional state, g) study, including going to school and doing homework and h) contact with other people. The individual interviews were aided by 16 pictures (negative and positive pictures for each performance). If children reported an impact on any performance, the frequency of the impact and the severity of its effect on their daily life were scored. Children were also asked to identify oral problems that in their opinion, caused the impact. The oral problems were identified from the list complied in the first step of the assessment.The oral impact score of each performance is obtained by multiplying severity and frequency scores, 0, 1, 2 or 3 each, in relation to that performance. Therefore scores can range from 0 to 9 per performance. The overall impacts score is the sum of all 8 performances (ranging from 0 to 72) divided by 72 and multiplied by 100. An alternative method of reporting the severity of oral impacts, from the same data set, is to use the 'intensity' and 'extent' of impacts. The intensity refers to the most severe impacts on any of the 8 performances or the highest performance score. It is classified into 6 levels; none, very little, little, moderate, severe and very severe  completed all stages of the survey. 52.4% were male and 47.6% were female. Their mean age was 11.3 years (sd = 0.6). The highest percentage of their fathers were agricultural workers or labourers (34.5%), 30.5% worked in business/private, 27.5% in governmental sectors, 2.1% did not work and 5.4% of children did not have a male guardian. The highest percentage of mothers worked in business/private (38.6%), 24.5% in agriculture, 21.1% in governmental organisations. 14.9% did not work and 1.0% did not have a female guardian.This population had a low level of dental caries: 43.1% were caries free and the DMFT scores ranged from 0 to 12 with a median score of 1.0 and a mean of 1.5 (±1.8). Almost all children (97.0%) had a Community Periodontal Index (CPI) score of 1 or more; 84.2% had calculus. In terms of oral hygiene status, 5.4% had good, 69.1% had moderate and 25.5% had poor oral hygiene. OHI-S scores ranged from 0.5–5.5 with a median of 2.5 and mean score of 2.5 (±0.9), indicating a moderate level of oral hygiene.The prevalence of oral impacts was high; 89.8% of children had experienced some kind of oral impact on their daily life during the past three months. There was no difference between the prevalence of impacts in girls and boys (Chi-square test). Impacts on Eating were the most prevalent 72.9%). The prevalence of impacts on Emotion (58.1%), Cleaning teeth (48.5%) and Smiling (40.1%) were also relatively high. The remaining prevalences of impacts were lower, namely Study (15.4%), Relaxing (14.7%), Contact with people (12.2%) and Speaking (9.9%) (Table .9%. The Among the children with impacts, the extent of impacts varied from 1 to 8 performances with impacts (PWI); 16.2% had 1 PWI, 23.3% had 2, 26.9% had 3 and 18.4% had 4 PWIs. Few children had 5 or more PWIs. About 1 in 5 children had severe or very severe intensity of impacts; 18.7% had severe and 2.6% had very severe intensity of impacts.15.9% had very little, 31.1% had little and 31.7% had moderate intensity of impacts . This is surprising in that this was a low caries population in an area with a free accessible school dental service. Although there is no study using OHRQoL index with a population-based sample of 12 year olds to compare with, findings of previous OHRQoL studies suggest that oral impacts are very common in children of this age. In Brazilian adolescent populations, the prevalence of impacts was 32% ,20 and 69.8%. ThiDespite the fact that oral impacts were prevalent in this Thai child population, they were not severe. For example, half of this population had Child-OIDP score less than 7.6 and half of those with impacts had very little or little intensity of impacts Table . MoreoveThis study found that eating was the most important aspect of OHRQoL of children. Difficulty with eating due to oral problems was the most common impact (72.9%), and led to more severe oral impacts on children's quality of life than impacts on other performances. Oral ulcers and exfoliating primary teeth contributed to eating difficulties in nearly half of those with impacts. The finding that eating was the most common performance affected is similar to all studies using the OIDP in all age groups ,21-24. TDifficulty with smiling was another important aspect of children's OHRQoL. It affected 40% of children. The most prevalent cause was position of teeth. Dissatisfaction with position of teeth, moreover, accounted for oral impacts in 1 in 5 of all children Table . AlthougGum problems were the other important oral conditions affecting children's OHRQoL. More than one fifth of children perceived that bleeding and swollen gums caused oral impacts on their life, particularly in relation to difficulty cleaning, a problem experienced by nearly half of all children Table . ChildreAn interesting finding was that impacts relating to social dimensions, such as study being affected and contact with people, were less common and least severe. Schor suggested that children's social performances rely more on their physical and psychological performances than adults .It is apparent that an important reason for the high prevalence of oral impacts in children is natural processes such as exfoliating primary teeth or spaces due to a non-erupted permanent tooth. They contributed largely to the high incidence of impacts in these pre-adolescent children. On the other hand, these conditions were not reported as important causes of oral impacts in other age groups ,22. The Although children could often not specify precisely which impairments led to impacts, the question of perceived clinical causes should exclude impacts from some conditions which are definitely not related to actual impairments as well as to treatment needs. For example, toothache, ulcers and conditions relating to appearance definitely require different treatment and could be easily differentiated. However, the accuracy of detecting perceived impairment is limited in a population-based study, while it can be improved at the individual level of investigation.The specific age group under investigation, particularly in relation to their stage of development, may have influenced the high prevalence of oral impacts. Developmental changes unavoidably affect HRQoL between childhood and adolescence . MaturitThe prevalence of oral impacts on daily performances in this child population was very high. Oral impacts affected children's quality of life mainly through difficulty eating and smiling. There are various oral conditions that contributed significantly to the incidence of impacts, namely, sensitive teeth, toothache, oral ulcers and exfoliating primary teeth. Although the prevalence of impacts was high, the severity was not; many children had their quality of life affected at low levels. This reveals a need for further longitudinal studies to better understand and interpret OHRQoL measures in children.SG carried out all work including data collection, data analysis and writing the paper. GT supervised the project and assisted writing. AS initiated the idea of, and supervised the project and edited writing.


Recombinant growth hormone preparations have been examined by several methodologies. In this study recombinant human growth hormone ; deamidation of N149, and N152, and oxidation of M140, M125 and M170. Peak area comparison of the modified and parental peptides indicates that these changes were present in ~2% of the recombinant preparation.Electrospray LC-MS analysis revealed one major protein with an average molecular mass of 22126.8 Da and some additional minor components. Electrospray LC-MS/MS evaluation of the enzymatically digested Genotropin70 remains unclear.Modifications of the recombinant human growth hormone may lead to structural or conformational changes, modification of antigenicity and development of antibody formation in treated subjects. Amino acid exchanges may be caused by differences between human and E. coli codon usage and/or unknown copy editing mechanisms. While deamidation and oxidation can be assigned to processing events, the mechanism for possible di-methylation of K This single mutation causes autosomal dominant growth hormone deficiency type II by prolonged retention time of R183_H183 aggregates into secretory granules [The structural integrity of recombinant products generated by prokaryotic and eukaryotic organisms is a major concern. Modifications such as amino acid sequence substitution/mutations of recombinant proteins may lead to pharmacological inactivation, autoimmune phenomena -3 and ade-1 gene ,9. Zhu egranules .However, although such changes can be detrimental, non functional sequence alteration induced by poor editing of recombinant proteins may act as a marker of growth hormone abuse in situations such as athlete doping. We therefore were highly interested in the homogeneity and structure of rhGH preparations.® is expressed by E. coli, strain K12. It consists of a single polypeptide chain containing 191 amino acids and two disulfide bonds  [Genotropin82-C189)  with a mIn humans two major hGH splicing variants have been described, a 22 kDa protein and a 20 kDa protein, that bind different sites at the growth hormone receptor and serve different biological activities ,14.The genetic origin of hGH is the hGH-N gene, located on the long arm of chromosome 17, in a 66-kbp cluster region closely related to four other genes: hGH-V, hCS-A, hCS-B and hCS-L. The hGH-N gene is expressed in both, pituitary and several nonpituitary sites , all oth14 and M125 to polymerisation [A series of posttranslational modifications of hGH have been described and range from N-glycosylation, acetylation, deamidation, oxidation at Mrisation -18.® is expressed by E. coli. Since the fidelity of hGH translation in E. coli cannot rely on copy editing [® is of particular interest to for modern medicine and pharmacotherapy.As mentioned above, Genotropin editing ,20, nor  editing -23, ther®. This was achieved using two dimensional gel electrophoresis (2-DE), matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) followed by tandem mass spectrometry (MALDI-MS/MS) and liquid chromatography mass spectrometry (LC-MS) followed by tandem mass spectrometry (LC-MS/MS). These modern analytical tools provide definitive structural analysis independent of antibody availability and specificity.The aim of the present study was to investigate the homogeneity of a commercial available rhGH, Genotropin® showed a multiple spot pattern with masses between 20 000 and 35 000 Da and pIs from 4.5 to 7.0. Several two dimensional (2D) gels with sample amounts of 0.5, 1, 2, 5, 10, 20, 50, 100, 200 and 500 g of Genotropin® were performed. Decreasing protein load showed reduction of spot size and number and finally, limitation to two spots of 22000 Da with pI of 5.3 and 5.4.Two dimensional gel electrophoresis (2-DE) of 1 mg GenotropinNeither MALDI-TOF-TOF nor LC-MS analysis of picked gel spots indicated any modifications or isoforms in an amount, that would explain differences between the two spots.® have shown that the main product was a molecule with an average molecular weight (MW) of 22126.8 Da. The manufacturers had determined the average MW of Genotropin® to be 22124 Da.Electrospray liquid chromatography – mass spectrometry (LC-MS) measurements of the intact GenotropinThe mass difference of approximately 3 Da may originate from the deconvulation of some broader, lower intensity peaks. Several minor components could be also detected. Figure  The massElectrospray tandem liquid chromatography – mass spectrometry (LC-MS/MS) measurements of the samples prepared from one dimensional SDS-PAGE indicated mass differences at several peptides. Doubly or triply charged ions were chosen for all MS/MS experiments due to their better fragmentation pattern. Table 70 . According to the ratios of the peak areas of the peptides containing the unmodified and possibly substituted methionines, these changes were present at < 2% of the whole protein amount. A mass increase of 57 Da was detected at the peptide LFDNAMLR. It could be localized at the N terminus of the peptide and it is supposed to be an artefact of the alkylation step during sample preparation. All modifications were partial; in each case peptides with both modified and unmodified residues were present. LC-MS/MS spectra for all modified peptides are available as supplementary material.Deamidation of the amino acids N® and identified by MALDI-TOF on the basis of peptide mass matching [Approximately 96 spots were excised from a 2D gel with a sample load of 1 mg Gentotropinmatching  followin® was unambiguously identified by MS and MS/MS Data  resulted in the identification of amino acid substitutions at residues M14, M125 and M170. Di-methylation of K70 or exchange to arginine, deamidation of N149 and N152, and oxidation of M14, M125 and M170 were also observed. These sequence alteration account for 2% of the recombinant protein.The dominant protein in the Genotropin65_V65 and rhGH Q66_K66[14_W14) and wild type human growth hormone  with the result that the hGHv had more binding affinity to its receptor than hGHwt [14 influenced binding, even if it is not a "hot spot" for linkage to its receptor. Furthermore, the amino acid exchanges detected may very well lead to antigenic differences and thus form the molecular basis for eliciting immune responses.Amino acid exchanges of a rhGH has been described before: Gellerfors et al. (1990) describe exchanges rhGH QH Q66_K66. Since tan hGHwt ,27. Cunn® may have been generated as the base pair G-G / G-A was replacing G-C. Arginine (R)_K/Q and R_G exchanges of rhGH Genotropin® may be due to difficulties in translation of the rare codon AGG. Kane et al. (1995) predicted translational problems with an abundant mRNA species containing an excess of rare tRNA codons that may arise after the initiation of transcription of a cloned heterologous gene in the E. coli host [The underlying cause of amino acid exchanges may be codon usage and/or absence of copy editing in E. coli: The M_I exchanges may be due to miscast of the third nucleoside of the cognate anticodon at the so-called Wobble-position, i.e. switch cytosine to guanine/adenosine, a phenomenon described by Crick as "Wobble- hypothesis . Crick (oli host . Recent oli host ,29. Tranoli host ,29-31.®: translation errors due to usage of (1) the rare codon AGG in E. coli and (2) incorrect codon usage consisted with Crick's "Wobble-hypothesis".In summary, we found two different pathways for amino acid exchanges in Genotropin14 and M125 oxidation as detected by LC-MS [170 as in our study. Indeed Teh et al. (1987) oxidised natural hGH extracted from pituitary glands and detected M14 and M125 oxidation by reversed phase chromatography [170, as detected by reversed phase chromatography [Oxidative modification of a recombinant human growth hormone has been described by Karlsson et al. (1999) who demonstrated Mby LC-MS . No othetography . Gellerftography . It is w70 in rhGH and hGH have not previously been reported.Post translational modification such as N-acetylation, N-glycosylation, deamidation and oxidation have been reported for rhGH, hGH and bovine growth hormone (bGH) -17,34. D41, K70, and K115[70 in Genotropin® could have biological relevance, probably reducing its pharmacotherapeutic activity.Whether transmethylation occurred during processing or is a post translational event during rhGH production in E. coli is unknown. Nevertheless, Martal et al. (1985) demonstrated reduction of biological activity of hGH and bGH by methylation and ethylation of its residues K and K115. Therefo149 and N152 may be due to technical processing, probably by heat treatment or lyophilisation and has already been reported by Gellerfors et al (1990) and Karlsson et al. (1999) [Deamidation of N. (1999) ,32. Thou. (1999) ,27,36.Modifications of the recombinant human growth hormone, as shown in this study, may effect functionality and safety depending on the prevalence of such forms in the preparation. As already mentioned above, impaired binding to the receptor, conformational changes leading to impaired function, amino acid exchanges as mutations may well lead to immune phenomena or even disease -3,37. In®) including amino acid exchanges, oxidation, di-methylation and deamidation. This analytical battery is a reliable, specific and sensitive analytical tool for this purpose.Using one- and two-dimensional gel electrophoresis, electrospray LC-MS, LC- MS/MS and MALDI-TOF-TOF mass spectrometry we detected a series of modifications of the recombinant human growth hormone  was suspended in 0,5 ml of sample buffer consisting of 8 M urea (Merck, Darmstadt, Germany), 4% CHAPS (3- [(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate) (Sigma, St. Louis, MO, USA), 10 mM 1,4-dithioerythritol (Merck, Germany) and 0,5% carrier ampholytes "Resolyte" 3,5–10 (BDH Laboratory Supplies, Electran ®, England). The suspension was transferred into Ultrafree-4 centrifugal filter units (Millipore, Bedford, MA), for desalting and concentrating proteins. Protein content of the supernatant was quantified by the Bradford protein assay system [Genotropin y system . The staOne dimensional SDS-polyacrylamide gel was performed as described by Laemmli . Samples2 DE was performed essentially as reported . SamplesElectronic images of the gels were recorded using Adobe Photoshop and Microsoft Power Point Softwares.+ ions of angiotensin I, angiotensin II, substance P, bombesin, and adrenocorticotropic hormones (clip 1–17 and clip 18–39). Each spectrum was produced by accumulating data from 200 consecutive laser shots. Those samples which were analysed by PMF from MALDI-TOF were additionally analysed using LIFT-TOF/TOF MS/MS from the same target. A maximum of three precursor ions per sample were chosen for MS/MS analysis. In the TOF1 stage, all ions were accelerated to 8 kV under conditions promoting metastable fragmentation. After selection of jointly migrating parent and fragment ions in a timed ion gate, ions were lifted by 19 kV to high potential energy in the LIFT cell. After further acceleration of the fragment ions in the second ion source, their masses could be simultaneously analysed in the reflector with high sensitivity. PMF and LIFT spectra were interpreted with the Mascot software (Matrix Science Ltd, London, UK). Database searches, through Mascot, using combined PMF and MS/MS datasets were performed via BioTools 2.2 software (Bruker). A mass tolerance of 100 ppm and 2 missing cleavage sites for PMF and MS/MS tolerance of 0.5 Da and 1 missing cleavage sites for MS/MS search were allowed and oxidation of methionine residues was considered. The probability score calculated by the software was used as criterion for correct identification.Spots were excised with a spot picker , placed into 96-well microtiter plates and in-gel digestion and sample preparation for MALDI analysis were performed by an automated procedure  ,42. BrieThe algorithm used for determining the probability of a false positive match with a given mass spectrum is described elsewhere .® MiniQuick 0.6 mg  was suspended in the solution provided in the two-chamber cartridge and diluted with 1% formic acid  in water  to 1 pM/μl. 1 μl of this solution was used for the nano-electrospray LC-MS investigation. The HPLC used was an UltiMate™ system  equipped with a PepMap C18 purification column (300 μm × 5 mm) and a 75 μm × 150 mm analytical column of the same material. 0.1% TFA  was used on the Switchos module for the binding of the peptides and a linear gradient of acetonitrile  and 0.1% formic acid in water was used for the elution. The gradient was  0% B for 12 min, 80% B in 30 min, 100 % B in 3 min, 100% B for 10 min, 0% B in 2 min, 0% B for 23 min. The flow rate was 240 nl/min. The LC-system was coupled on-line to a QSTAR Pulsar hybrid mass spectrometer . The nanospray source of Proxeon (Odense, Denmark) was used with the distal coated silica nanospray capillaries of New Objective (Woburn, MA, USA). The electrospray voltage was set to 1800 V. Spectra were acquired over the mass range of m/z 600–1600. The accumulation time was 1 sec. Protein spectra were deconvoluted by Analyst® . LC-MS/MS analyses were carried out also with the UltiMate™ system interfaced to the QSTAR Pulsar or to an LTQ  linear ion trap mass spectrometer. The gradient was (A = 5% acetonitrile / 0.1% formic acid in water B = 80% acetonitrile / 0.1% formic acid in water) 0% B for 12 min, 60% B in 88 min, 100 % B in 5 min, 100% B for 10 min, 0% B in 5 min, 0% B for 20 min. Peptide spectra were recorded over the mass range of m/z 450–1300, MS/MS spectra were recorded in information dependent data acquisition over the mass range of m/z 50–1600. One peptide spectrum was recorded followed by two MS/MS spectra on the QSTAR Pulsar instrument; the accumulation time was 1 sec for peptide spectra and 2 sec for MS/MS spectra. The collision energy was set automatically according to the mass and charge state of the peptides chosen for fragmentation. One full spectrum was recorded followed by 3 MS/MS spectra on the LTQ instrument, automatic gain control was applied and the collision energy was set to the arbitrary value of 35. Doubly or triply charged ions were selected for product ion spectra. MS/MS spectra were interpreted by Mascot (Matrix Science Ltd, London, UK).GenotropinThe author(s) declare that they have no competing interests.Felizardo, Maureen carried out MALDI-TOF-TOF analysis. Raja, Karlin participated in sequence alignments and in the design of the study. All authors read and approved the final manuscript.


Human resources are the most important assets of any health system, and health workforce problems have for decades limited the efficiency and quality of Latin America health systems. World Bank-led reforms aimed at increasing equity, efficiency, quality of care and user satisfaction did not attempt to resolve the human resources problems that had been identified in multiple health sector assessments. However, the two most important reform policies – decentralization and privatization – have had a negative impact on the conditions of employment and prompted opposition from organized professionals and unions. In several countries of the region, the workforce became the most important obstacle to successful reform.This article is based on fieldwork and a review of the literature. It discusses the reasons that led health workers to oppose reform; the institutional and legal constraints to implementing reform as originally designed; the mismatch between the types of personnel needed for reform and the availability of professionals; the deficiencies of the reform implementation process; and the regulatory weaknesses of the region.The discussion presents workforce strategies that the reforms could have included to achieve the intended goals, and the need to take into account the values and political realities of the countries. The authors suggest that autochthonous solutions are more likely to succeed than solutions imported from the outside. Health reforms that aim at increasing efficiency, quality and users' satisfaction need to take into consideration human resource issues, because the health sector is labor-intensive and the performance of health systems depends on qualified and motivated workers -4. At thIn Latin America, the need to improve the performance of the workforce had been pointed out in many health sector assessments conducted in the 1970s and 1980s by the United States Agency for International Development (USAID), the World Bank (WB), other agencies and independent researchers.  were too often guided by favoritism, political dictates, and nepotism;• Health professionals were insufficiently committed to the public system due to the conflict of interests mentioned above, poor personnel management systems and the perception that wages were low;• The medical profession strongly dominated the definition of health sector policies and the regulation of the conditions of practice of all health professions;• Communication between providers and patients was poor, and providers and service users had very different social and cultural backgrounds. In countries with Amerindian-speaking populations, providers did not speak their languages;• The regulation of training institutions and conditions of practice was weak;• The training of health promoters and other auxiliary personnel such as dental assistants, midwives, laboratory technicians, equipment maintenance and repair technicians, and pharmacy clerks was poor or non-existent, thus their performance was poor.According to the literature reviewed, these conditions led to low productivity and efficiency; inadequate equipment; shortages of supplies and drugs; unmotivated and inadequately trained staff; questionable quality of care; and low users' satisfaction.By the mid-1970s, the need to reform the human component of the health services was very urgent, and the urgency increased with the severe economic downturn that countries of the region suffered during in the early 1980s. The size of the Latin America health labor force (about nine million ) impliedThis paper reviews the impact of the health reforms carried out under the leadership of the World Bank. Data come from a review of the literature including the leading Latin American and non-Latin American journals, monographs, documents found in ministries and reform offices, technical reports, papers presented at conferences and fieldwork carried out by the authors between 1980 and the present in Bolivia, Colombia, Costa Rica, Dominican Republic, Ecuador, El Salvador, Honduras, Mexico and Peru.In the early 1980s, with a few exceptions, countries in the region entered a severe economic downturn. The International Monetary Fund and the World Bank required – as a condition for new lending and for refinancing the debts – reduction of public spending. The impact of structural adjustment programs on health services was severe and compromised the ability of governments to maintain the physical infrastructure of the facilities, provide the necessary supplies and equipment, and maintain competitive salaries for the workforce.The World Bank took advantage of this situation to provide loans to the ministries of health and social security funds. Together with the loans, the Bank offered guidelines for the reorganization of the health services according to the ideological economic principles held by the institution.The World Bank had started its activities in the health sector in the early 1970s, and by the early 1990s it was the world's largest health sector lender. In 1999, the total accumulated value of health, nutrition, and population loans worldwide amounted to USD 16.8 billion (in 1996 dollars) . By the The underlying principles of the Bank-led reforms include the belief that the private sector is more efficient than the public sector, and that decentralized administrative units are better-equipped to respond to the needs of the population than centralized governments. The Bank also proposes to limit government financing to a basic package of services, to be determined for each country by cost-effectiveness studies and the country's ability to pay. Services included in the basic package are made available at no cost to the indigent population; governments and the rest of the population subsidize them. In the Bank's health reform model, the role of the state is limited to that of a regulator of the health care market. The United States health system is closer to the World Bank model than the national health services or social security funds of other industrial nations.The World Bank-led reforms did not include strategies to correct the human resources problems presented earlier, even if some of them had been identified by Bank-sponsored studies; the World Bank reformers believed that market forces would resolve them. As we will see, the contrary occurred and privatization and decentralization had some negative consequences for the workforce. The reliance on the market also hid the structural problems that needed to be taken into account at the time reformers designed human resource policies. The design and implementation process which were characterized by secrecy, generated dissatisfaction among health workers.We have organized our analysis of human resources during the reforms under five categories: resistance of the workforce to the implementation of a market-oriented health care model; faulty implementation processes; inadequately trained personnel in managerial positions and in service delivery; institutional and legal dimensions including insufficient and faulty information, lack of financial resources, and civil service statutes; and weak regulatory legislation to ensure the quality of professionals and the performance of the sector.The neoliberal health reforms intended to change the values that had inspired the Latin American systems and the relations between the government and the health workforce. Thus, health would no longer be considered a right; only the insured would be entitled to receive a broad array of services. Health workers would lose the protection they had enjoyed as public servants and become part of a flexible workforce Table .Humans tend to resist change, but opposition to health reform was compounded by conflicts with the value system that inspired it. Most Latin American constitutions recognize health as a right and most governments had adopted Alma Ata's primary health care principles as their strategy to promote Health for All. Health policies were based on the principle of solidarity and fostered cooperation among health workers and also between them and other workers in related sectors such as education and agriculture . It was Human resource changes were needed to implement Alma Ata, but the budget reductions required by the World Bank caused a significant deterioration of working conditions. As their purchasing power worsened, health workers intensified undesirable behaviors to increase their income. These included levying illegal fees, diverting patients from the public sector to their private clinics , using pAs indicated, Latin American physicians have supplemented their income from the public system by means of their private practice. Physicians value the stability and fringe benefits of a public post, but as the public delivery system deteriorated in many countries, private clients became their main source of revenue. By the late 1980s in most Latin American countries , more than half of the health expenditures, including the cost of medicines, occurred in the private sector , and in One of the objectives of the neoliberal reforms was to create a more flexible labor force by decreasing the number of tenured public employees and increasing the number of temporary personnel. This change threatened: the job security of the civil service, a very important dimension in countries with little political stability and where workers are frequently exposed to arbitrary political removals; the providers' income, which now would depend on their ability to compete for contracts and clients; and that which workers expected from their employer . The promoters of health reforms failed to acknowledge that in a politically unstable region, civil service tenure was necessary to maintain an efficient, productive, and loyal labor force. Predictably, these threats triggered the opposition of professional associations and unions, led to strikes, and lowered productivity during the reform process.Health workers of most countries of the region are unionized . The uniLabor unions anticipated that the decentralization and privatization of the sector would have a negative impact on their membership and their bargaining power. Labor unions in Bolivia, Ecuador, El Salvador, Mexico, Nicaragua, Peru and Venezuela expressed their opposition to the reforms on the grounds that they were an excuse for governments to relinquish their constitutional responsibility to ensure access to care and would lead to dramatic changes in work conditions . In someAnalysts of the health reform policies agree with the workers' concerns. Segall  asserts In addition, health professionals strongly opposed the idea of having their professional autonomy limited by professional administrators who would force them to adhere to diagnostic and treatment protocols based on economic principles rather than on technical criteria. The cool reception that Mexican professional associations offered to foreign insurance companies and health maintenance organizations is a good example of this concern .The large majority of countries in Latin America do not have accurate information on the numbers and distribution of the health workforce. There is no centralized entity responsible for gathering information on the health personnel practicing in the country or in the different geographical regions, and even when only public sector workers are examined, the numbers collected at different administrative levels differ . The reforms worsened the possibility of having accurate information. If the system is decentralized and the majority of providers are in the private sector, the sources of information from which human resources data will need to be collated multiplies. The challenge may be even greater if each agency gathers the information differently, and without this basic information it is very difficult to engage in human resources development planning Table .Civil service laws and negotiated agreements with worker unions have limited the freedom of public sector managers to reorganize the workforce; managers had to "administer the rules"  issued eManagers overcame these constraints by hiring additional personnel under flexible contracts. In Brazil there are now about 15 different types of contracts for public sector employees; a significant expansion of the use of temporary workers and contracts without fringe benefits has been reported in Argentina , ColombiTransferring human resources to other politico-administrative levels was more difficult than anticipated. Kolehmainen-Aitken  and HomeDecentralizing human resources is expensive. Prior to decentralization – in countries such as Bolivia, Colombia and Mexico – local or regional governments frequently hired additional personnel under different pay scales and benefit packages than those used by the central government. After decentralization, each decentralized administrative unit had to set pay scales, fringe benefits, performance assessments and reward systems for its workers. Because it was not possible to lower the salaries and reduce the benefits of workers with higher salaries and benefits – generally federal/national workers – it was necessary to bring the salaries and benefits of all the workers to the level of those who had the most generous package. Higher salaries and benefits represent an increase in the fixed costs of the system for an indefinite period.Moreover, the creation and/or strengthening of the decentralized administrative structures are often not accompanied by a decrease in the number of federal employees, further raising the costs of personnel to the system . In MexiDecentralization has been promoted under the assumption that the proximity of decision makers to the communities facilitates providing services more in accordance with the needs of the community. But the decentralized structures' ability to respond to local needs has been constrained by: the civil servants they inherited, who are often inadequate in terms of skill mix and geographical distribution; the conditions of employment imposed by other government sectors and the unions; and the local elite, who do not have in mind the health needs of the communities and who lobby to have relatives and friends hired by local health authorities.In several countries of the region, decentralization has broadened the urban-rural inequities in the distribution of personnel and in the quality of services -54. RichCountries considered the human resource implications of reform only when they faced resistance from the unions or realized they did not have the financial and human resources required to implement the reform . The heaThe human resources units of the health ministries and the regional and local administrations were inadequately prepared for the reform Table . TraditiThe tasks required by the reform overstretched the capacity of these units. Included among these tasks were: the development of new organizational structures, defining new job descriptions, reassigning responsibilities and designing new reporting systems, establishing performance evaluation methods and assisting all decentralized units to carry out similar duties in their jurisdictions.The decentralization of personnel often unveils rivalries and discontent among personnel who feel unfairly treated. All these issues need to be addressed, negotiated and resolved; and most human resources units were ill-prepared to lead the process . For exaThe mismatch between the abilities of the workforce and the needs of the system documented in the 1980s still prevails. The lack of coordination between training institutions and employers is at the root of the problem, along with weaknesses in the regulation of health professions and the dominance of physician's groups on the policy making process.According to Bach , shortagIn most countries, managerial positions were traditionally given to physicians with little or no management training . The neoThe promoters of the reforms recognized the need for good managers but, because they had heavily criticized the public sector and questioned its role, it was difficult to recruit qualified staff into managerial positions and, in turn, it was difficult for those recruited to motivate and retain qualified staff ,57. MostThe Pan American Health Organization  evaluateAfter having spent millions of dollars in training and infrastructure, the region's capacity to manage contracts is still limited , and wheCountries in the region used a top-down approach to define and implement reform. The implementation was often led by a handful of top health executives, newly created reform offices, the political elite and international agencies, which in turn contracted for the technical assistance of international consultants and universities closely aligned with the neoliberal ideology of the World Bank. In general, there was little interest in involving professional associations, unions or even local universities in this process ,56 Tabl.In Costa Rica the labor unions were involved initially in the reform, but their influence was undermined by the World Bank and the Inter-American Development Bank, which adopted a more closed, centralized decision-making style . AccordiSome authors have offered a different interpretation of the exclusion of the workforce and assert that it was the complexity of the human resources issues and the need to involve many players  in their solutions that led reform promoters to ignore the labor force and other stakeholders .Regardless of the underlying motivation, the secrecy that surrounded the process of defining and implementing the reforms produced rumors, confusion and hostility against the reform among civil servants and professional groups ,39,67,68In addition, health reformers did not consider the strategies and resources needed during the transition and early stages of implementation; such as allocating new financial resources and establishing clear communication channels. In addition, as discussed in the previous section, the reforms failed to adequately train managers who could lead the transition to a new management system .The need for regulation increases in health systems where the private sector plays a prominent role. In Latin America it was not until the early 1990s, largely as a consequence of the health reforms, that health policy makers became aware of the urgency to regulate the health system. A regulatory system includes adequate regulations, the institutions to enforce them, and a judicial system that ensures a timely response in the event of conflict. Enforcing regulations is important to guarantee that trained personnel provide safe and adequate services Table .Prior to the reforms, the regulatory systems were poorly developed and, when they existed, they were not tailored to the needs of consumers and enforcement was very limited . For insThe number of medical schools has grown spectacularly in the last two decades and, in most countries, there were no mechanisms to ensure the quality of the training institutions or to test the abilities of the graduates. The number of medical schools in Chile grew by 68% between 1992 and 2000, in Peru the growth was also by 68%, in Argentina by 61% and in Brazil by 21%. The growth occurred mainly in the private sector . Costa RBy its very nature the regulation of the health profession relies heavily on the opinions of professionals and especially on physicians, who in turn place a great value on their autonomy and have had little interest in responding to social and political demands . MedicalThe dominance of physicians has alienated other professions such as therapists, nurses, pharmacists, optometrists and psychologists . For exaThe regulation of the health professions has a long way to go in Latin America, and it is probably impossible to establish sustainable regulatory mechanisms in the absence of political and judicial reforms; for the system to work, it needs to free itself from political interference . Some arBach , Brito eThe implementation of the reforms has been uneven in the Latin American region. Technical, logistical, political and financial problems have surfaced everywhere. While most countries decentralized, a few – such as Colombia and Chile – managed to significantly expand private insurance and, with the exception of Brazil, very few have engaged in large contracts with private sector providers. The most salient feature has been significant changes in hiring modalities. In Brazil there are 15 different types of contract arrangements  and in PAnother important result of the reform is the surge of multiple jobs, particularly in Argentina, Brazil, El Salvador, Panama, Peru, Uruguay, and to a lesser degree Chile ; that haA survey of nurses conducted in Argentina, Brazil, Colombia and Mexico  revealedFurthermore, according to the Tavistock Group  "cooperaSimilarly, one of the basic principles of neoliberal reforms – that the efficiency of the system will increase by using flexible contracts and rewarding productivity – is not supported by the data. The World Bank conducted an evaluation of civil service reforms in 15 countries and concluded: "None of the cases reviewed so far have revealed any empirical evidence that the Civil Service Reform and related Technical Assistance Loans have succeeded in fostering the needed change in work attitudes, ethics and organizational culture that could lead to greater efficiency/productivity in the civil service" [Research has also uncovered problems with using performance-based payment schemes. For example, in health it is often difficult to establish who is responsible for the outcome. Costa Rica implemented a pilot project in Barva de Heredia in which physicians received an incentive based on productivity, but this was not extended to the nurses and the other clinic staff . The proHealth providers can also manipulate the information to maximize their benefits rather than the well-being of the patients; and substandard working conditions rather than workers' action may be responsible for a poor outcome . MexicanEstablishing a valid and reliable merit-pay system is extremely complicated; placing too much emphasis on material rewards may displace more intrinsic motivators such as the pleasure of doing good, or caring for the patient. Bennet and Franco  even sugThere is a belief among neoliberal economists that private sector workers are more productive and less corrupt than public employees. Recent hospital studies confirm that because of fear of termination, absenteeism is less frequent among non-tenured physicians hired through short-term contracts than among civil servants, but short-term contracts have not increased commitment to the institution . CorruptDecentralization can also be seen as a transfer of financial responsibilities from central government to local authorities, which has the potential to affect wages and job stability  and incrIn decentralized health systems, particular attention needs to be paid to establishing good coordination among those responsible for the vertical program at the central level, the decentralized administrative units, and the clinical staff. The absence of good coordination may result in health workers' reporting to two supervisors: the person responsible for the vertical program and the supervisor of the health facility or region, as is the case in Mexico .In sum, in the majority of Latin American countries, the neoliberal reforms have not made the health delivery model more responsive to the needs of the community; have not increased the productivity of health workers; have had a negative impact on working conditions and staff motivation; appear to have further compromised the quality of care; and have had a limited impact on the capacity to regulate the health professions and training institutions.More recent studies suggest that many of the old workforce problems remain unresolved ,70,86,87Health reform provided a perfect opportunity to promote and encourage workforce improvement. Prerequisites for the progress of such processes are political will, effective relationships between the educational and service-providing institutions, and the open collaboration of professional groups. However, the reforms had the opposite effect. The neoliberal orientation challenged the use of conventional regulation strategies because, by encouraging professionals to seek their own interest instead of the interests of society as a whole, it questioned whether society and regulatory bodies could continue to trust and have faith in the criteria expressed by health professionals.Human resources account for the lion's share of health budgets, and poor performance had been identified as the main constraint to efficiency, quality of services and user's satisfaction. The values that guided the neoliberal reform and the privatization and decentralization initiatives worsened the problems affecting the Latin American workforce and added new challenges. The reform implementation process was also responsible for the failures. The promoters of the neoliberal health reforms underestimated the importance of involving professional organizations and unions in the planning and implementation of the reform efforts, raising suspicion and resistance among organized health workers. In addition, the reforms were designed in secret and implemented using a top-down approach. The only significant advance in human resource development in the last 10 years is the increased interest in strengthening the capacity to regulate training institutions and practitioners.Solving the problems that affect human resources is no easy task, and probably the solutions are different for each country; ignoring the problems and hoping that the market will resolve them is a recipe for failure. Managing change is very complex; embarking on reform without having secured the collaboration of the workforce and ensuring the availability of sufficient qualified staff is irresponsible.The exclusion of the organized labor force from reform discussions is indefensible; the collaboration and motivation of the health workers is essential to the reform, and the leaders of the organized groups can assist in informing and aligning the workforce ,89.Policy changes requiring a different skill mix of health personnel require careful planning because there is a significant time lag between deciding that there is a need to train additional professionals and having them available. For example, a 10% rise in the number of students in a medical school produces only a 2% increase in the supply of doctors 10 years later . Most coOne thing that reforms could have done was to support interventions that, independently from the reform, some countries had designed to overcome workforce weaknesses. Some interventions were national programs, others were pilot projects, and there were also experimental projects. The following are examples of these autochthonous interventions.To solve the urban-rural gap and improve equity, most ministries of health in the region created the obligatory rural health service known as pasantía or servicio social that requires physicians to spend one year in a rural health center prior to graduation or immediately after receiving their degree .A few social security institutes have also found ways to reduce health inequities; such is the case of the Mexican and Ecuadorian Institutes of Social Security (IMSS and IESS). IMSS organized COPLAMAR, an extensive primary and secondary care program for poor rural populations, which brought general practitioners and specialists to rural areas. The Ecuadorian program known as Seguro Social Campesino offered primary care services for rural dwellers and, when needed, hospitalization at the Ecuadorian Social Security Institute; this program aimed at reducing the rural-urban gap in a country of which 70% of the population then lived in the rural areas .Mexico created a training program for traditional midwives who worked in dispersed rural populations; the objective of the program was to enhance the quality of their services and reduce maternal and infant mortality . The MinCosta Rica attracted the world's attention with the Open Walls Hospital, a program that required the specialists of a regional hospital to schedule – when needed – weekly visits to dispersed rural populations. The program also intended to convey the message to specialists that they were not different from other health workers and had an obligation to serve poor rural dwellers even when doing so would involve personal inconvenience .To enhance the professional status of primary care practitioners, reduce referrals, and improve quality of care, IMSS created many positions in the specialty of family physician, forcing the medical profession to recognize the status of the new specialty. Colombia's Ministry of Health sent nurses to a graduate health education program taking advantage of a US fellowship program in order to diversify the human resource composition of the Ministry.Some of these projects were successful; this was the case of family physicians and COPLAMAR in Mexico. However, the first 14 Mexican states that decentralized in the 1980s dismantled the program, transferring it to the states' departments of health to create the state health system, and the quality of rural health deteriorated rapidly .Others function poorly, as is the case of the compulsory year of social service in all the countries where it was established. Similarly, the health promoters program in the Dominican Republic suffered from insufficient training, lack of continuous education, absence of efficient supervision, and poor remuneration, comments that can be extended to all health promoter programs of the region. The Seguro Social Campesino has suffered from inadequate financing, and plans to extend it to the entire rural population have been placed on permanent hold.Other programs were discontinued because of indifference and lack of support from policy makers. Thus, the Hospital without Walls ceased after all Ministry of Health hospitals were transferred to the Social Security Institute. Due to budgetary problems, the Colombian Ministry of Health did not employ the health educators on their return from graduate school.The health reforms would have provided a perfect opportunity to support and strengthen many of these and other autochthonous interventions. A proper course of action would have been to evaluate the projects that had been developed locally, identify their strengths and weaknesses, and establish their viability and sustainability. Through trial and error and with appropriate resources and incentives, many of them are likely to be more effective and less costly than foreign programs invented by those who hardly know the realities of developing countries and are inspired by ideological principles and questionable economic theories.There is much more that needs to be done to improve the training and management of human resources for health, and very often the solutions depend on the collaboration of a wide range of stakeholders such as those who produce health workers, those who employ them, those who pay for their services, those who negotiate working conditions and those who define the standards of professional practice. It is no easy task and can be successfully accomplished only if there is strong political will, if there is openness and trust among all stakeholders, and if sufficient resources and time are allocated to this effort. Most countries of the region have the capacity to find appropriate solutions to the problems they are facing. Dussault  argued tAs Segall mentioned , it is iThe author(s) declare that they have no competing interests.Both authors have contributed equally to the design, data collection, data analysis, drafting and completion of this article.


Most lymphomas that involve the tonsil are large B cell lymphomas. Large B-cell lymphoma is a high grade malignancy which progresses rapidly. Tonsillar lymphoma usually presents as either a unilaterally enlarged palatine tonsil or as an ulcerative and fungating lesion over the tonsillar area. Small lymphocytic lymphomas (SLL) of the Waldeyer's ring are uncommon.We report a 41-year-old male who presented with a ten-year history of snoring. Physical examination revealed smooth bilateral symmetrically enlarged tonsils without abnormal surface change or cervical lymphadenopathy. Palatal redundancy and a narrowed oropharyngeal airway were also noted. The respiratory disturbance index (RDI) was 66 per hour, and severe obstruction sleep apnea (OSA) was suspected. No B symptoms, sore throat, odynophagia or dysphagia was found. We performed uvulopalatopharyngoplasty (UPPP) and pathological examination revealed incidental small B-cell lymphocytic lymphoma (SLL).It is uncommon for lymphoma to initially present as OSA. SLL is an indolent malignancy and is not easy to detect in the early stage. We conclude that SLL may be a contributing factor of OSA in the present case. Adenotonsillar enlargement is the main cause of obstructive sleep apnea (OSA) in the pediatric population. However, this prevalent syndrome is more complicated in adults . OSA has2, 91%, minimal SaO2, 62%, and a desaturation index (≥ 4%) of 61.8/h. The arousal index was 64.8/h and the respiratory disturbance index (RDI) was 66.0/h. Believing that the patient was suffering from severe OSA and hyperplastic palatine tonsils, he received UPPP.A 41-year-old man presented with complaints of snoring, excessive daytime sleepiness, and pavor nocturnes for more than 10 years. Systemic diseases were denied. Physical examination revealed bilateral symmetric and enlarged palatine tonsils without abnormal surface change. There were no palpable cervical lymph nodes or B symptoms (fever, body weight loss and cold sweats). White and red blood cell counts, biochemistry and chest radiographs were within normal limits. The results of an overnight polysomnography (PSG) showed mean SaOThe postoperative course was uneventful and sleep apnea improved. PSG performed 4 months after surgery demonstrated that the RDI had reduced to 23.9/h. Pathology indicated small B cell lymphocytic lymphoma Figure ,2 with bAdenotonsillar enlargement is the leading cause of OSA in the pediatric population  though iSmall lymphocytic lymphoma (SLL) is an indolent but relentless malignancy, with a median survival of about 10 years. Because It usually presents as neck lymphadenopathy in the later stages, SLL is not easy to diagnose in the early stage. The effectiveness of chemotherapy for treating SLL is controversial. Most studies have found no benefit in treating patients until they develop symptoms . Lymphomet al., [et al., [Nolan described a case of adenotonsillar enlargement due to chronic lymphatic leukemia which caused severe OSA . His repet al.,  reportedet al., . Abe et [et al.,  describe[et al., .Tonsillar surgery should be performed even on patients highly suspected of having lymphoma to improve OSA -10. NeckNone declared.YT, YC, CL, WC and MT made substantial contributions to the intellectual content of the paper, in the interpretation of results and in drafting the manuscript. All authors read and approved the manuscript


The control of proliferation, differentiation and survival of normal and malignant cells in the tumour microenvironment is under the control of a wide range of different factors, including cell:cell interactions, cytokines, growth factors and hormonal influences. However, the ways in which these factors interact are poorly understood. In order to compare the effects of multiple variables, experimental design becomes complex and difficult to manage. We have therefore evaluated the use of a novel approach to multifactorial experimental design, the Taguchi methods, to approach this problem.The Taguchi methods are widely used by quality engineering scientists to compare the effects of multiple variables, together with their interactions, with a simple and manageable experimental design. In order to evaluate these methods, we have used a simple and robust system to compare a traditional experimental design with the Taguchi Methods. The effect of G-CSF, GM-CSF, IL3 and M-CSF on daunorubicin mediated cytotoxicity in K562 cells was measured using the MTT assay.Both methods demonstrated that the same combination of growth factors at the same concentrations minimised daunorubicin cytotoxicity in this assay.These findings demonstrate that Taguchi methods may be a valuable tool for the investigation of the interactions of multiple variables in the tumour microenvironment. The control of proliferation, differentiation and survival of normal and malignant cells is under the control of a wide range of different factors. These include cell:cell interactions, immune regulatory factors, hormonal influences, and local environmental influences. However, the way in which these factors interact to regulate the dynamics of the malignant cell population are poorly understood. It is important to identify important factors and the way that they interact in order to rationalise treatment and develop new therapeutic options. However, one of the main problems is the difficulty in designing experiments to compare the effects and interactions of multiple variables. For example, a traditional experimental design to compare seven independent variables at three different concentrations each requires a large number of individual experiments (2187 experiments). The logistical and resource implications of this experimental design make these experiments very difficult to carry out. We have investigated the use of an alternative approach to experimental design, the Taguchi Methods , baculovWe have therefore used a simple and reproducible assay, the MTT assay, to evaluate whether the Taguchi methods can be used to investigate the effect of G-CSF, GM-CSF, IL3 and M-CSF on daunorubicin mediated cytotoxicity in K562 cells.Taguchi methods consist of 3 phases: designing the experiment, running and analysing, and confirming and validating the assumptions. After selecting the variables to be studied, Taguchi methods depend on distributing the factors under study in an orthogonal array, which distributes the variables (factors) in a balanced manner. Examining a typical orthogonal array Table , where eA(BC), the subscript L, which is designated by A, represents the number of experiments that would be conducted using this design, B denotes the number of levels or concentrations within each column which denotes how many levels or concentrations could be investigated, while the letter C identifies the number of columns available within the orthogonal array which indicates how many factors or variables could be included in the experiment [8(27) means that 8 experimental runs are needed to investigate 7 different factors, each of which is set at 2 predetermined levels or concentrations  is simply the range, which could be calculated as the difference between the highest and lowest average response of each level. For example the impact of factor A on the experiment outcome is the difference between A1 & A2. ). The effects of all factors are calculated in the same way, then arranged in a response table, and examined for those factors with the strongest effect (i.e. highest difference Δ), in order to separate them from the weak effects. The breaking point between the strong and weak effects is identified as a change in the pattern of the difference between the ranges around the median.Besides determining the effects of the individual factors, the same technique is used to determine the strength of the impact of interactions on the product of the experiment. The calculations are performed as the previous section. In order to determine the interactions between A and B, the average result of each 2 factors combined must be determined. This is achieved through calculating the values of 4 points: A1B1, A1B2, A2B1, and A2B2, where A1B1 is the average result generated due to the interaction between concentration 1 of both factors, A1B2 is the result of the interaction between concentration 1 of factor A and concentration 2 of factor B, A2B1 represents the interaction between factor A at concentration 2 and factor B at concentration 1, while the fourth point A2B2 is the interaction between both factors at concentration 2. These 4 points are then presented graphically to show the strength or weakness of the interaction. Whether the interaction is weak, mild, or strong depends on whether the two response lines are parallel, converging or intersecting, with intersecting lines indicate a strong interaction, and parallel lines indicate no interactions. Once the strong factors and interactions have been identified, an estimate of their combined effect is calculated and the new experiment is designed according to these assumptions. An experiment is then carried out – referred here to as "confirmation run"- to validate the assumptions upon which the new experiment was based. Conducting a confirmation run and the comparison between the actual and the predicted results is necessary. If however, the confirmation results are disappointing, the planning phase must be re-evaluated and the elements that went into the experiment must be reviewed. A possible cause could be the omission of a key factor from the experiment, for example a powerful interaction was not considered. Another common cause is the setting of factor levels too close together for the experiment. In these situations, the factor is found insignificant during the analysis and is not accounted for in the validation. The confirmation run should include the best or preferred settings for mild and weak influences as well as the strong ones. However, the less influential factors are not incorporated into the prediction equation. The reasoning is that the differences in the average results may be due to experimental variation, and to incorporate their effects could result in an overestimate of the predicted results. This could lead to a disappointing confirmation run when actually the results would have validated the experiment analysis if the predicted results had not been artificially high or low.2-95% air. Cells were plated in 96 well microtiter plates (200 μl) at a density of 3 × 104 cells/ml. Cells were co-cultured in the presence of 0.1 μg/ml daunorubicin.K562 cells were cultured in RPMI-1640 medium supplemented by 10% (v/v) foetal bovine serum, 50 μg/ml penicillin and 25 μg/ml streptomycin at 37°C in a humidified atmosphere of 5% COIn all experiments K562 cells were co-incubated in the presence or absence of cytokines concentrations shown in Table 50 μl of MTT (3–4,5-dimethylthiazol 2,5-diphenyl tetrazolium bromide) (5 mg/ml) was then added to each well and incubated at 37°C for 4 hours. The resulting deep blue crystals were dissolved in 0.04 N HCl Isopropyl alcohol, and the absorbance measured using a scanning multiwell spectrophotometer at dual wavelength 570–630 nm. All measurements were performed in triplicates.The % survival was calculated asIn classical experimental design the effect of each factor, each concentration and each interaction is tested independently. In order to investigate the full interactions between 4 factors each at 2 concentrations, requires 81 individual experimental conditions to be performed. This study used 49 combinations. The structure of the 49 experiments is shown in Table 8(27) to investigate the effect of 4 cytokines on the survival of K562 leukaemic cells. This array accommodated 4 factors MCSF, IL-3, GMCSF and GCSF. The interaction between MCSF and the other cytokines was inserted into the array. As the 3rd column was used to examine the interaction between MCSF and IL-3, columns 5 and 6 were used to assess the interaction between the same cytokine and GMCSF and GCSF respectively  was observed8 series , four points were computed A1D1, A1D2, A2D1, and A2D2 Table .A1D1 is the average result of the combined effect of MCSF at a concentration 100 U/ml and GCSF at a concentration of 10 ng/ml.The interaction matrix AXD was then represented graphically Fig  which shAn estimate of the predicted response (μ) based on the selected levels was then computed. The calculations were based on the overall average value (T) and the effect that each of the recommended levels of the strong factors and interactions has on the overall average.μ = T+(A2D2 - T)+(B2 - T)+(A2C2 - T)+(A2B2 - T)-(A2 - T)-(A2 - T)-(A2 - T)-(B2 -T)-(C2-T).The reason for subtracting the individual effects of factors A, B, and C from the effects of A2B2 is that A2B2 is comprised of the effects of factor A, factor B and the interaction itself. Unless the effects of the two factors are subtracted these strong effects would be included twice and resulting in an overestimation of the predicted result.The predicted survival derived from the above prediction equation was 43%. A confirmation run that produces a %survival close to 43%would validate the assumptions of this Taguchi method. The actual confirmation run, in fact, resulted in 39.84% survival rate indicating the success of the Taguchi experiment.Further calculations were performed to determine whether the outcome of other combinations could be predicted from the Taguchi experiment and confirmed by analysis. The results are shown in Table The aim of this study was to evaluate the ability of the Taguchi methods to investigate the effects of several factors simultaneously on the death and/or survival of the malignant cells, and to compare this strategy against a traditional full experimental design.A major finding of the study was that the Taguchi methods predicted the combination of factors that results in the lowest survival of the malignant cells. This agreed with the conclusions of the full experimental design but required only eight individual experiments to pinpoint this combination. However, it must be stressed that the Taguchi methods are not intended to be a replacement for traditional experimental design, but if used as a complimentary strategy can make analysis of complex interactions feasible and practical. In this study, for example, eight individual experiments produced a testable combination which required 49 individual experiments to produce in the traditional experimental design. In more complex systems, only Taguchi methods become feasible. For example, to study 13 factors at 3 different combinations would require 1,594,323 individual experiments at a cost for re agents alone of over 27 million pounds. If Taguchi methods are used, this can be reduced to just 27 individual experiments at a cost of under 500 pounds.in vivo, and we propose that the Taguchi methods may be a useful strategy to understand these interactions in vitro, and to help devise and implement new therapeutic strategies.We have described a novel experimental approach to studying the interactions of several factors on the cytotoxicity of malignant cells. We show that the method is effective in the determination of the optimum conditions, even in the presence of multiple interactions. We anticipate that this experimental strategy will have many applications in the investigation of complex interactions. For example we have used this strategy to model the complex testicular microenvironment and the ability to support the survival of acute lymphoblastic leukaemia cells (manuscript in preparation). These sort of interactions are common in the survival of malignant cells None declared.HM designed the Taguchi assay, carried out cell survival assays, and drafted the manuscript. KLY participated in the design and coordination, and produced the final manuscript. APJ conceived the study and participated in its design and coordination. All authors read and approved the final manuscript.


Identification of effective systemic antineoplastic drugs against anaplastic thyroid carcinomas has particularly important implications. In fact, the efficacy of the chemotherapeutic agents presently used in these tumours, is strongly limited by their low therapeutic index.In this study gemcitabine was entrapped within a pegylated liposomal delivery system to improve the drug antitumoral activity, thus exploiting the possibility to reduce doses to be administered in cancer therapy. The cytotoxic effects of free or liposome-entrapped gemcitabine was evaluated against a human thyroid tumour cell line. ARO cells, derived from a thyroid anaplastic carcinoma, were exposed to different concentrations of the drug. Liposomes formulations were made up of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/cholesterol/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-MPEG (8:3:1 molar ratio). Cell viability was assessed by both trypan bleu dye exclusion assay and fluorimetric analysis of cell DNA content.A cytotoxic effect of free gemcitabine was present only after 72 h incubation . When gemcitabine was encapsulated in liposomes, a significant effect was observed by using lower concentrations of the drug  and earlier exposure time (24 h).in vitro against human thyroid cancer cells, the gemcitabine incorporation within liposomes enhances the drug cytotoxic effect with respect to free gemcitabine, thus suggesting a more effective drug uptake inside the cells. This may allow the use of new formulations with lower dosages (side effect free) for the treatment of anaplastic human thyroid tumours.These findings show that,  At present, the prognosis of anaplastic thyroid carcinomas is very poor ,2. SinceGemcitabine is a new fluorinated nucleoside analogue provided with a potent anti-tumour activity, tested in vitro and in vivo against a variety of solid malignancies -6. In ad1-difluorodeoxycytidine) hydrochloride (HPLC purity >99%) was a kind gift of Eli-Lilly Italia S.p.A.  and was used without further purification. Cholesterol was obtained from Sigma Chemicals Co. (St. Louis, USA). 1,2-dipalmitoyl-sn-glycero-3-phosphocholine monohydrate and N-(carbonyl-methoxypolyethyleneglycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (MPEG-2000-DSPE) were purchased from Genzyme products (Suffolk, United Kingdom). Double-distilled pyrogen-free water from Sifra S.p.A.  was used. Sterile saline was a product of Frekenius Kabi Potenza S.r.l. . All other materials and solvents were of analytical grade .Gemcitabine (2,2Liposome formulations were made up of DPPC/Chol/MPEG-2000-DSPE (8:3:1 molar ratio). To maximize the entrapment efficiency of a hydrophilic molecule such as gemcitabine, various liposome preparation procedures ,12 were y = 6.958 × 10-3 + 0.3971x, where y is the UV adsorbance and x is the drug concentration) presented a r2 value of 0.9993. The amount of gemcitabine entrapped in liposomes was determined as a difference between the amount of drug added during liposome preparation and the amount of the entrapped drug present in the supernatant. The encapsulation efficiency was expressed as percentage of the total amount of gemcitabine that became entrapped.The loading capacity of liposomes was evaluated after removing the free gemcitabine (centrifugation at 14000 × g for 1 h at 4°C) from the vesicular colloidal suspension coming from the re-suspension of freeze-dried liposomes. The amount of gemcitabine in the supernatant was spectrophotometrically determined at 268.8 nm (Shimadzu UV-1601). UV calibration straight-line  before they reach their target site, one of these is the large volume of distribution after i.v. administration. This situation elicits a low therapeutic index and a high toxicity in healthy tissues . After eIn the last years, gemcitabine has emerged as a very potent anti-tumour drug and it is currently used, alone or in combination, in the treatment of patients with different malignancies, including ovarian, pancreatic, non-small cell lung and other cancers -6. Despi®.In this study, we prepared a liposome formulation with a high entrapment efficiency of gemcitabine and tested its efficacy against a well established human poorly differentiated thyroid carcinoma cell line. Our data demonstrate that, in ARO cells, incorporation of gemcitabine in liposomes enhances the cytotoxic effect of the drug as compared to free gemcitabine, suggesting a more effective uptake of the drug inside the cells. This activity was demonstrated at concentrations even lower than serum levels obtained in clinical trials. In our knowledge, no data are available in the literature concerning the relationship between gemcitabine uptake and its cytotoxicity, since neither liposomes or other carrier system have been tested against tumour cells. Our preliminary experiments (unpublished observations) show that a similar effect (improved uptake and cytotoxicity at lower doses) may be obtained also in colon cancer cells. It is note worthing that the various component used for the preparation of the liposomal delivery device were already approved by regulatory offices to be used in the preparation of liposome-based formulations, i.e. DoxilThe clinical predictive value of the in vitro cell line preclinical cancer model is well established  and the None declared.The pre-publication history for this paper can be accessed here: