The habitual "any other comments" general open question at the end of structured questionnaires has the potential to increase response rates, elaborate responses to closed questions, and allow respondents to identify new issues not captured in the closed questions. However, we believe that many researchers have collected such data and failed to analyse or present it.General open questions at the end of structured questionnaires can present a problem because of their uncomfortable status of being strictly neither qualitative nor quantitative data, the consequent lack of clarity around how to analyse and report them, and the time and expertise needed to do so. We suggest that the value of these questions can be optimised if researchers start with a clear understanding of the type of data they wish to generate from such a question, and employ an appropriate strategy when designing the study. The intention can be to generate depth data or 'stories' from purposively defined groups of respondents for qualitative analysis, or to produce quantifiable data, representative of the population sampled, as a 'safety net' to identify issues which might complement the closed questions.We encourage researchers to consider developing a more strategic use of general open questions at the end of structured questionnaires. This may optimise the quality of the data and the analysis, reduce dilemmas regarding whether and how to analyse such data, and result in a more ethical approach to making best use of the data which respondents kindly provide. The survey is a key method in health services research . The majThere are four types of questions which might require an open rather than closed response , and the amount they actually write. Thus researchers may be able to determine the status of a general open question at the design stage of a study by having a strategy to generate depth and treat the data qualitatively, or by having a strategy to generate shorter responses as a 'safety net' for complementary or new issues. Having such a strategy may help researchers to devise a strategy for analysis and publication.Researchers can determine the status of a general open question at the design stage of a study by having a strategy to generate depth and treat the data qualitatively. For example, at the end of a structured questionnaire about use of Chinese medicine, one researcher invited respondents to tell the 'story' of their use of Chinese medicine, leaving a full one and a half pages of white space, and offering a example of the detail required. The following instructions were given: 'Now tell us your own story, using the space on the next page. We've provided one true patient story to give you an idea of the kinds of details we need. The important subjects are repeated in the list above the space we've provided for you to write in. Also use the back of the page if you wish. Please remember to write clearly.' . This apIn the above example, the 80% response suggests that the stories obtained could be viewed as representative of the population surveyed. However, in qualitative research the validity of the study does not rest on the researcher's ability to demonstrate representativeness with respect to the total population. Rather, it rests on transferability whereby the researcher offers detailed description of the setting in which the research was undertaken . Thus whIf the open question is used to generate qualitative data, then researchers will need to use qualitative analysis techniques and possibly qualitative software as used in the Chinese medicine example discussed previously , and wilGeneral open questions may produce little more than the closed questions on the questionnaire  and rathFrom a quantitative perspective, the strength of a survey approach is representativeness, and thus non-response bias should be a concern. Respondents are less likely to complete a general open question than a closed one on a postal questionnaire : 81% of Those who choose to answer the general open question could be different from respondents overall, either being more articulate or having a greater interest in the survey topic. It is important to consider and report on who has made written comments so that bias can be considered. In a patient satisfaction survey, females were more likely to make comments than males but interestingly there were no significant differences by age group or educational status . In a suFormal analysis must be rigorous so that the findings are useful and convincing. Content analysis may be undertaken ,3, where1. Reads a sub-set of the comments.2. Devises a coding frame to describe the thematic content of the comments.3. Assigns the codes to all the comments. The coding frame can be applied using software designed for this purpose  or manua4. The codes can be entered into a statistical package alongside the data from the closed questions and treated as variables in a quantitative analysis.The coding process is time consuming and requires expertise ,4,6. TheWhen reporting the responses to general open questions it is important that the numbers of respondents making each comment are displayed, with recognition that although a specific number of people mentioned an issue it might be relevant to many more who did not choose to mention it. Although numbers rather than percentages tend to be used when reporting responses to open questions ,12,15,26Publication of responses to open questions can occur within a paper reporting the main findings of the questionnaire or as a separate publication. Where comments elaborate and explain findings from closed questions, it may be most appropriate to publish them in the same paper . Where aAn explicit strategy requires that researchers consider the role of a general open question in the context of their survey, and its status in terms of generating either qualitative or quantifiable data. If the role of the question is to give a voice to participants then the researcher can ensure that comments will be read to identify any concerns and queries expressed by individuals, and that appropriate action is taken with individual comments. If the role is to generate qualitative data then attention can be paid to generating depth of data and the quality issues associated with qualitative research. If the role is to act as a safety net and generate quantifiable data then resources will need to be allocated for reading the comments, and if there appears to be added value, for formally analysing the data with attention to non-response bias and reliability of coding. Having a strategy may reduce any dilemma faced by researchers about whether and how to analyse these questions, may help the researcher to allocate the appropriate time and expertise to this data, and may produce an analysis robust enough for publication in peer reviewed journals. A potential disadvantage of having such a strategy may be that some flexibility is lost and that some important issues are missed. Finally, researchers cannot assume that they know how best to facilitate a respondent to complete a general open question and may need to consider using cognitive aspects of survey methodology to construct the question .• General open questions at the end of structured questionnaires can present a problem to researchers who may face the dilemma of whether or not to analyse them.• They are necessary when piloting questionnaires because they identify further issues for inclusion in the survey, and may be a bonus in the main study because they may increase response rates and may identify issues which complement responses to closed questions.• The value of such questions, and the quality of the data and analysis, may be optimised if researchers make more strategic use of them by being clear about their role, and understanding the type of data they wish to generate when they design their study.• An explicit strategy for generating qualitative data will encourage attention to depth of data and issues important to the analysis of qualitative data such as reflexivity.• An explicit strategy for generating quantifiable 'safety net' data, that is important issues missed by the closed questions, will encourage attention to non-response bias and reliability of coding.The author(s) declare that they have no competing interests.AOC conceived the paper and wrote the first draft. KJT reviewed it critically, and developed the sections on qualitative research. Both authors produced the final draft and read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The treatment guidelines for tuberculosis treatment under Directly Observed Treatment, Short-course (DOTS) have been a common strategy for TB treatment in Zambia. The study was carried out in Ndola, Zambia, to investigate factors contributing to treatment non-adherence and knowledge of TB transmission among patients on TB treatment, in order to design a community-based intervention, that would promote compliance.A household-based survey was conducted in six randomly selected catchment areas of Ndola, where 400 out of 736 patients receiving TB treatment within the six months period, were recruited through the District's Health Management Board (DHMB) clinics. All patients were interviewed using a pre-tested structured questionnaire, consisting of i. Socio-demographic characteristics ii. Socio-economic factors iii. Knowledge about TB transmission and prevention iv. Patterns in health seeking behaviour and v. TB treatment practices at household level.Most male TB patient respondents tended to be older and more educated than the female TB patient respondents. Overall, 29.8% of the patients stopped taking their medication. There were 39.1% of the females and 33.9% of the males, who reported that TB patients stopped taking their medication within the first 2 months of commencing treatment. Age, marital status and educational levels were not significantly associated with compliance. The major factors leading to non-compliance included patients beginning to feel better (45.1% and 38.6%), lack of knowledge on the benefits of completing a course (25.7%), running out of drugs at home (25.4%) and TB drugs too strong (20.1% and 20.2%). There was a significant difference  in TB knowledge, with more males than females reporting sharing of cups as a means for TB transmission, after adjusting for age, marital status and educational levels. Significantly [p = 0.016] more patients who had resided in the study for less than two years (59%) were more likely to report mother to child transmission of TB, compared to 41.2% of those who had been in the area for more than 2 years.This study established that 29.8% of TB patients failed to comply with TB drug taking regimen once they started feeling better. Tuberculosis is one of the major public health risk factors of high mortality among patients in Zambia. The prevalence of tuberculosis among adults has more than doubled since the onset of AIDS epidemic in Zambia , with moAlthough tuberculosis treatment guidelines had been circulated for the management of the disease at health facility down to household level, DOTS was the only best means of increasing compliance among TB drug-taking patients in Zambia ,7. As eaWork carried out in Haiti , Zambia Issues of diverse nature to caring of patients at home are rampant in communities that have low literacy levels ,14. The Discrimination on the basis of disease at health facilities sometimes exacerbates problems with compliance with tuberculosis drug taking behaviour . Unless More often than not, when patients are diagnosed as having TB, communities immediately construct them as social and sexual misfits in the society, which is often followed by exclusion from social interactions and relationships . In homeA cross sectional study was conducted among tuberculosis patients who were getting their drugs from the clinics operational in Ndola, Zambia by the Ndola District Health Management Board. In this study, compliance refers to patients who took their TB drugs daily for 8 months. On the other hand, any patient who stopped taking TB drugs during the treatment period was regarded as non-compliant patient. This study took into account an analytical approach of comparing two groups (the compliant against the non-compliant group). The study was limited to these patients because of the research focus on compliance of patients over time to prescribed TB drug regimens identified from medical records. In addition to medical records, TB patients were also directly asked if they had ever stopped taking the drugs since starting the treatment.Using the medical records from the six sampled Ndola District Health Management Board Clinics, a list of all TB patients seeking treatment within the six months period was drawn. A total of 736 patients were listed from the six study sites. Using this figure, and a prevalence of 50% +_5% at 95% confidence level, the minimum sample size was determined to be 248. Although 400 TB patients were recruited in the study, 18 were excluded from the analysis because only demographic data was obtained. Reasons for incompleting questionnaires included some patients becoming restless and rushed to the hospital, while others were too sick to continue with the interview and had died by the re-appointment date.TB patients were classified as follows: those who never attended the TB-clinic after diagnosis, those who attended 1–2 times after diagnosis and commencement on treatment, and those who attended regularly (i.e. 3 or more times) after diagnosis and continuation of treatment. Patients who attended the clinic regularly and continued taking the treatment were classified as controls, and the rest of the patients were classified as cases.Out of 18 clinics that were run by the District Health Management Board in Ndola, six were randomly selected from the list of clinics so as to enable the team obtain a representative sample of the clinics in the district. In the sampled clinic catchment areas, a household-based survey was conducted and all patients that met the criteria were invited to take part in the study. In all, 114 non-compliant and 268 treatment-compliant TB patients participated in the study.TB is strongly associated with HIV/AIDS because of the perceived mode of transmission, which stigmatises and discriminates patients. These are common in most African communities  and ZambThose who had agreed to participate were interviewed using a pre-tested structured survey instrument. Data collected were individual socio-demographic characteristics and economic characteristics, knowledge on TB transmission, prevention and treatment practices, patterns in health seeking behaviour, compliance and non-compliance to TB drug taking, and perceived community support during the drug taking period.The data was entered in EPI6. It was edited using consistency and range checks after data entry. However, data analysis was done in the statistical package, SPSS. The Chi-squared Yates corrected test for 2 by 2 tables and Pearson's uncorrected Chi-squared test for higher contingency tables were used to determine associations between qualitative factors. A multivariate logistic regression model was used to adjust for differences in the distributions of age, marital status and education between males and females in the relationships between knowledge items and gender. The cut off point for statistical significance was set at the 5% level.The demographic characteristics of household-based patient population that participated in the survey are presented in Table Table No significant differences by duration of residence in the community were observed in knowledge of TB transmission through sexual intercourse, sleeping in the same room with TB patient and patient coughing directly at others Table . SignifiA total of 114 (29.8%) out of 382 patients stopped taking TB drugs at some point during the treatment regimen. Analyses for the factors associated with TB drug compliance revealed that sex, education, marital status, sharing a room with any one else, relationship with head of household, anyone suffered from TB in the house, close relative or friend that has suffered from TB, number of times suffered from TB, and use of traditional healers for treatment of TB were not significantly associated with compliance.The common reason given for stopping treatment by both the compliant and the non-compliant patients were that they could not continue with the medication when they started feeling well (45.1% and 38.6%), respectively. Meanwhile, other reasons given by complaint patients were lack of knowledge on the benefits of completing TB course (25.7%), TB drugs too strong (20.1%) and lack of food in the home (11.4%). Similarly, the non-compliant patients mentioned running out of drugs at home (25.4%), TB drugs too strong (20.2%) and loss of hope to live (16.7%) as reasons for stopping.Common reasons given for stopping treatment for male patients were that they ran out of drugs at home (29.1%) or had no food (23.6%). Meanwhile, most females reported that forgetting to take the medicine (32.0%), reaction to drugs (20.0%), and running out of drugs at home (20.0%) as the reasons for stopping taking drugs Table .Results from the qualitative study reveal that 10 out of 17 non-compliant patients stressed running out of drugs as a reason for stopping. A young man who was a widower aged 32 years reported:Okay, the drugs got finished but like I am not feeling well, that is why I don't go to collect, because here, there are no children you send. My nephew also has started what? The one who used to get it for me [referring to the nephew who had started work]In another in-depth interview with a single non-compliant female patient aged 31 years, emphasised on running out of drugs at home and reported thus:That one we drink two tablets, I..., we went on Monday, they said the Doctor was not there [referring to the clinical officer or nurse responsible for running the TB clinic]... he comes on Wednesday, so I have missed also.The key determinant for stopping medication in both qualitative and quantitative was running out of drugs.th month of consistently taking medication, more men tended to stop taking their medicine . About a third of the respondents  indicated that they stopped taking drugs within the first two months of starting treatment. However, by the 5The Ndola study showed that 29.8% of all TB patients on treatment did not comply. The majority of patients who participated in the study were between 20 and 39 years. This population is in agreement with other studies that had been conducted in Ndola ,5,24. ItIt has been demonstrated in other studies ,26 that No relationship has been found between marital status of an individual and the knowledge of TB. Lack of knowledge makes TB a serious public health problem, which needed an urgent attention. TB patients in Ndola could have continued sharing a room with an infected person, because of lack of accomodation, high rental costs and inadequate knowledge on TB transmission. Over 70% of TB patients easily mentioned the symptoms which included sweating at night, loss of weight, loss of appetite and prolonged coughing, which at times was accompanied by sign of blood in the sputum. Knowledge about the symptoms of the disease did not vary according to levels of education or age.Knowledge modes of transmission and methods of prevention required revisiting, as community members seemed to have had knowledge that did not relate the disease to the environment (that TB could be transmitted through the air). They believed in physical contacts with objects, for instance, sharing of cups, having sexual intercourse with TB patient and from mother to child. This result is consistent with findings from a study in Botswana , which rIt is not surprising therefore, to find no gender differences in terms of TB knowledge transmission among the age groups. The low knowledge in preventive measures in this group could be explained through our study sample itself. Although these age groups were of patients who could have received the information from the clinic during the time they were collecting drugs and their food supplies, which were often provided to patients, prevention did not seem to have been stressed by health care providers. The other minor reason could be that our questions were prompted; this could also explain why 13.3% reported that to prevent TB one should always keep to one partner. The care seeking behaviour remained the same as in other studies ,26, wherThe patients' duration of residence in the city of Ndola compared very well with another study , which fHowever, the study had identified some misconceptions on TB transmission, about a third of the respondents reported having got the diseases through sexual intercourse. This study suggested a relationship between community constructions of TB and HIV/AIDS campaigns. Patients could have related TB with HIV and selected sexual intercourse as a source of transmission for the disease, and therefore considered condom use as a means for preventing it. One explanation for this misconception could be attributed to the overstressing that TB is an opportunistic disease for HIV/AIDS. The study in Haiti , clearlyNon-compliance to TB drug taking is reasonably lower than we originally thought. There were 29.8% of the patients who stopped taking their medicine at two months after commencing treatment. The defaulting progressed in the second month (16.8%), and then stabilized thereafter. Factors associated with defaulting in the current study, included patients beginning to feel better, lack of knowledge on the benefits of completing a course, running out of drugs at home and TB drugs being too strong to continue. However, the major and stricking determinant of non-complaince was the patient beginning to feel better.While lack of food and other provisions were identified in other studies , as factOver 79% of the patients, had suffered from TB only once, while the rest had suffered more than twice. Results present a wide range in the percentage of opposing views for TB as an air borne disease. Within age groups, there was usually no relationship between the documented preventive measures against TB and air borne germs. The reasons were attributed to the fact that these were patients obtaining their drugs from a health facility, hence, received some of the information on disease transmission. The relationship between the awareness 'coughing blood' and 'TB' was explored during the health talks.The findings in the Ndola study showed that approximately 39.8% of all TB patients on treatment did not adhere to their treatment schedules, when they started feeling better.The author(s) declare that they have no competing interests.FADK was the principal investigator of the study and was responsible for the design, implementation and supervised data entry and cleaning. He worked closely with the biostatistician during data analysis. He is the principal author of this paper. MT was the co-investigator of the study. She contributed to the design of the study, coordinated data collection, entry and cleaning. She was part of the data analysis team. SS is a co-author of this puplication who carried out data analysis as a biostatistician. LS co-authored this publication and was responsible for data entry and cleaning. He was part of the data analysis team. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The major psychoactive cannabinoid compound of marijuana, delta-9 tetrahydrocannabinol (THC), has been shown to modulate immune responses and lymphocyte function. After primary infection the viral DNA genome of gamma herpesviruses persists in lymphoid cell nuclei in a latent episomal circular form. In response to extracellular signals, the latent virus can be activated, which leads to production of infectious virus progeny. Therefore, we evaluated the potential effects of THC on gamma herpesvirus replication.Tissue cultures infected with various gamma herpesviruses were cultured in the presence of increasing concentrations of THC and the amount of viral DNA or infectious virus yield was compared to those of control cultures. The effect of THC on Kaposi's Sarcoma Associated Herpesvirus (KSHV) and Epstein-Barr virus (EBV) replication was measured by the Gardella method and replication of herpesvirus saimiri (HVS) of monkeys, murine gamma herpesvirus 68 (MHV 68), and herpes simplex type 1 (HSV-1) was measured by yield reduction assays. Inhibition of the immediate early ORF 50 gene promoter activity was measured by the dual luciferase method.in vitro. Importantly, concentrations of THC that inhibit virus replication of gamma herpesviruses have no effect on cell growth or HSV-1 replication, indicating selectivity. THC was shown to selectively inhibit the immediate early ORF 50 gene promoter of KSHV and MHV 68.Micromolar concentrations of THC inhibit KSHV and EBV reactivation in virus infected/immortalized B cells. THC also strongly inhibits lytic replication of MHV 68 and HVS THC specifically targets viral and/or cellular mechanisms required for replication and possibly shared by these gamma herpesviruses, and the endocannabinoid system is possibly involved in regulating gamma herpesvirus latency and lytic replication. The immediate early gene ORF 50 promoter activity was specifically inhibited by THC. These studies may also provide the foundation for the development of antiviral strategies utilizing non-psychoactive derivatives of THC. The Kaposi's Sarcoma-Associated Herpesvirus (KSHV/HHV-8) is the likely cause of Kaposi's sarcoma in AIDS patients, aging individuals and organ transplant patients . KSHV iReactivation of latent virus is often initiated by extracellular signals activating through receptors and various transcription factors. Transcription factors activate the promoter of the critical viral gene open reading frame 50 (ORF 50) of KSHV, HVS, MHV 68 or its homologue Rta of EBV . The ORF9 tetrahydrocannabinol (THC), has been shown to modulate and primarily suppress immune responses against various pathogens . THCview see -26. CB1 view see ,18]. ThiSince THC is an immune modulator we hypothesized that it may have an effect on gamma herpesvirus replication and/or latency. The data presented show that THC inhibits reactivation and lytic replication of these herpesviruses, possibly through inhibition of the ORF 50 promoter.The KSHV positive primary effusion lymphoma cell line BCBL-1 isolated by Don Ganem and co-workers , and BC-Cell suspensions were loaded in wells of a vertical agarose gel as described ,32. The NIH3T12 cells were infected with MHV 68, OMK cells with HVS, and both cell types with HSV-1 at a multiplicity of infection of 2 in the presence or absence of various concentrations of THC dissolved in DMSO. Control cultures were treated with DMSO. IC50 was determined by measuring virus titers in THC treated and control samples after one cycle of freeze-thawing of the cultures. Additional controls were uninfected cells grown in the presence of THC or DMSO. THC or DMSO was present throughout the experiment.To evaluate the effect of THC on the KSHV ORF 50 promoter, a DNA fragment corresponding to the promoter region upstream of the mapped mRNAs [reference , nucleot50) of THC at 1 μg/ml or about 3.3 μM.To evaluate the effect of THC on KSHV replication, we selected BCBL-1 and BC-3 lymphoblastoid cells because these cultures spontaneously produce small amounts of virus, and we used a previously published antiviral drug testing protocol  to deterTo evaluate whether another KSHV strain is also susceptible to THC, the BC-3 cell line was also tested. In these cells, THC had a similar inhibitory effect on KSHV reactivation as observed with BCBL-1 cells (not shown).50) for THC was estimated at around 0.9 μg/ml or about 3 μM.Two experiments analogous to the one described for KSHV were performed with the EBV transformed B cell line P3HR1. P3HR1 cell cultures were grown in the presence of the phorbol ester TPA, because EBV did not reactivate spontaneously and TPA was essential to induce lytic EBV replication. The cultures were also supplemented with either various concentrations of THC dissolved in DMSO or with equivalent volumes of DMSO. Fig. We examined the effects of THC on the replication of MHV68 and HVS, two rhadinoviruses related to KSHV and to lesser extent to EBV. These viruses can infect monolayer cells and are suitable for testing the effects of compounds on lytic virus replication by virus yield reduction assays, a more widely used test for evaluating antiviral drugs. NIH3T12 cells were infected with MHV68 at a multiplicity of infection of 2 in the presence or absence of various concentrations of THC dissolved in DMSO. Control cultures were treated with DMSO. THC or DMSO was present throughout the experiment. Cell cultures were incubated for 48 h. As shown in Figure 50) was estimated at around 0.6 μg/ml, equivalent to about 1.9 μM. Similar results were obtained from two independent experiments. Similar and reproducible results were obtained with HVS in monkey kidney cells (not shown).To quantitatively determine antiviral effects of THC, yield reduction assays were performed. NIH3T12 cell cultures were infected with MHV 68 and incubated with THC or DMSO dilutions for 48 h. Cell-associated virus was liberated by freeze-thawing. After low speed centrifugation, virus titer was determined in the culture supernatants. Figure Because the observed effects might be due to a non-specific toxic effect of THC, we tested whether THC alters cell division or morphology in NIH3T12 and owl monkey kidney (OMK) cells. Monolayers of these cells were prepared at about 25% confluency and cultured for 2 days in the presence of THC concentrations ranging 0.6–10 μg/ml. A series of photographs Figure  show thaThe effect of higher THC concentrations on cell division was also determined. Exponentially growing BCBL-1 or NIH12 cells were cultured for two days in the presence of various concentrations of THC or DMSO and cell counts were determined. The 50% cell division inhibitory concentration of THC for BCBL-1 cells was around 33 μM. NIH3T12 cells were less sensitive and the 50% inhibitory concentration was around 99 μM (not shown).We next examined whether or not the THC inhibitory effect can be observed against other herpesviruses and tested whether drug treatment suppresses the replication of the alpha herpesvirus, HSV-1. Monolayers of NIH3T12 were prepared, infected with about 100 infectious units of HSV-1, and cultured for 3 days in the presence of THC concentrations ranging 0.6–10 μg/ml. A series of photographs shows Figure  that typWe also examined the effects of THC on production of HSV-1 virus yield in NIH3T12 cells. Cells were infected with HSV-1 at a multiplicity of infection of 2 in the presence or absence of various concentrations of THC dissolved in DMSO. Control cultures were treated with DMSO. THC or DMSO was present throughout the experiment. Cell cultures were incubated for 24 h and cell-associated virus was liberated by freeze-thawing. After low speed centrifugation the HSV-1 titer was determined in the culture supernatants. Figure To evaluate the effect of THC on the MHV 68 and KSHV ORF 50 promoters, luciferase reporter assays were performed. To assay the effect of THC on the MHV 68 gene, a DNA construct published by the Speck laboratory ,47 contaTo investigate whether the observed antiviral effects of THC are mediated through the cannabinoid receptors, two antagonists of THC, SR141716A (which acts on CB1) and SR144528 , were tested in the standard BCBL-1 KSHV reactivation assay  of four known antiviral drugs and of THC. Data on the antiviral effects of acyclovir, PFA and ganciclovir on KSHV were obtained from our Gardella gel assays [in vitro experiments; however, this drug is known to cause serious side effects and is toxic to the kidney in humans.To summarize the antiviral effects of THC and to compare THC with well-characterized antiviral drugs, we compiled data from the literature as well as from our own experimental results. Table l assays . Usherwol assays  also usel assays  using stAs outlined earlier, THC can modulate and inhibit the activation of immune cells, so it is not entirely surprising that it can down-regulate the reactivation of viruses residing in lymphocytes, as shown by the data. However, the antiviral effect of THC is not cell specific, since MHV 68 and HVS replication was also strongly inhibited by THC in NIH3T12 or owl monkey kidney monolayer cell cultures. This observation suggests that THC either directly or indirectly targets a viral gene shared by these herpesviruses.The data presented in Figure Early studies have attempted to evaluate whether THC has effects on HSV-1 and HSV-2 replication -44. MostInterestingly, statistical analysis indicates a lower incidence of Kaposi's sarcoma in HIV positive women using non-intravenous drugs . About 5We believe that studies on cannabinoids and herpesviruses are important to continue because there are obvious potential benefits. Better understanding may lead to the development of specific non-psychoactive drugs that may inhibit reactivation of oncogenic herpesviruses.None declared.MMM carried out most of these studies. TAS carried out some of the luciferase assays. TWK and HF participated in the design of the study. PGM conceived the study and participated in its design and overall coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Modulation of the immune system by genetically modified lymphoma cell vaccines is of potential therapeutic value in the treatment of B cell lymphoma. However, the anti-tumor effect of any single immunogene transfer has so far been limited. Combination treatment of recombinant IL-2 and IL-12 has been reported to be synergistic for inducing anti-tumor responses in solid tumors but the potential of IL-2/IL-12 gene modified B cell lymphoma cells has not been explored yet.Using three different human B cell lymphoma cell lines and primary samples from patients with B cell neoplasms, expression levels of the coxsackie B-adenovirus receptor (CAR) and alpha (v) integrins were analyzed by fluorescence-activated cell sorter (FACS). Adenoviral transduction efficiencies were determined by GFP expression analysis and IL-2 and IL-12 cytokine production was quantified by enzyme-linked immunosorbent (ELISA) assays. Proliferative activities of peripheral blood mononuclear cells (PBMC) stimulated with either cytokine derived from supernatants of transduced lymphoma cells were measured by cell proliferation (MTT) assays. An EuTDA cytotoxicity assay was used to compare cytotoxic activities of IL-2 and/or IL-12 stimulated PBMC against unmodified lymphoma cells.We found that B cell lymphoma cell lines could be transduced with much higher efficiency than primary tumor samples, which appeared to correlate with the expression of CAR. Adenoviral-expressed IL-2 and IL-12 similarly led to dose-dependent increases in proliferation rates of PBMC obtained from healthy donors. IL-2 and/or IL-12 transduced lymphoma cells were co-cultured with PBMC, which were assayed for their cytolytic activity against unmodified lymphoma cells. We found that IL-2 stimulated PBMC elicited a significant anti-tumor effect but not the combined effect of IL-2/IL-12 or IL-12 alone.This study demonstrates that the generation of recombinant adenovirus modified lymphoma cell vaccines based on lymphoma cell lines expressing IL-2 and IL-12 cytokine genes is technically feasible, induces increases in proliferation rates and cytotoxic activity of co-cultured PBMC, and warrants further development for the treatment of lymphoma patients in the future. Lymphoma cells are attractive targets for gene transfer, because these cells are potentially susceptible to immunotherapeutic strategies . Among tInterleukin-2 (IL-2) and interleukin-12 (IL-12) are cytokines that elicit strong antitumor effects by stimulating immune cells, including T cells and natural killer (NK) cells. Although either cytokine stimulates the proliferation of T cells, the production of interferon-γ (IFN-γ) by NK cells, and ultimately the cytolytic activity, the magnitude, and the spectrum of stimulatory effects by IL-2 and IL-12 are different. Although IL-2 is a stronger stimulator of proliferation and cytolytic activity, IL-12 is a stronger inducer of IFN-γ from NK cells and activated T cells. Although the combination of recombinant IL-2 and IL-12 treatment has been reported to be synergistic for inducing anti-tumor responses, systemic administration of these cytokines causes toxic side effects. Recent reports of intra-tumoral co-injection of adenoviral vectors expressing IL-2 and IL-12 demonstrated the regression of pre-established solid tumors with high frequency . HoweverRecently, we described an adenoviral protocol accomplishing highly efficient gene transfer to B-lymphoma cell lines . The useIn the present study, we have evaluated the relationship between the amount of cytokine production by the combination IL-2 and IL-12 and the in vitro effective anti-tumor activity. Using three different human B cell lymphoma cell lines and primary samples from patients with B cell neoplasms, we transduced both IL-2 and IL-12 genes by adenoviral vectors, and monitored cytokine production and effects on proliferation and cytolytic activity of co-cultured human peripheral blood mononuclear cells (PBMC).2 at 37°C. Virus propagation was performed in the Ad5 E1-transformed human embryonic retina cell line 911 [The following cell lines were analyzed: Raji , Braunschweig, Germany), Daudi , and OCI-Ly8-LAM53 . The cell lines were grown in RPMI 1640 with Glutamax  supplemented with 10% heat-inactivated fetal calf serum (FCS) , 50 μg/ml streptomycin, and 50 μg/ml penicillin (PAA), and were kept in a humified incubator with 5% CO2 and subcultured every 3 days in fresh complete medium and IL-2.Non-adherent Ficoll-Hypaque  separated human PBMC were obtained from whole blood from healthy donors and maintained in RPMI 1640 with Glutamax (Life Technologies) supplemented with 10% FCS (PAA), 50 μg/ml streptomycin, and 50 μg/ml penicillin. Cytokine-induced killer (CIK) cells were generated as described previously ,9. In br2 and could be maintained in culture for 10–12 days.Five patients diagnosed with lymphoma were included into this study; four patients with chronic B cell lymphocytic leukemia (B-CLL) and one patient with immunocytoma (IC). After informed consent, peripheral blood was obtained and lymphoma cells were isolated by Ficoll-Hypaque (Seromed) density centrifugation. Cell surface antigens were analyzed for the expression of CD19, integrin avβ3, integrin avβ5, and the coxsackie B-adenovirus receptor (CAR). Primary cultures were maintained in liquid culture in RPMI 1640 with Glutamax (Life Technologies) supplemented with 10% heat-inactivated FCS, 50 μg/ml streptomycin, and 50 μg/ml penicillin at 37°C, 5% CO5 cells in 50 μl of PBS plus 1 mM MgCl2/1% HS, at different multiplicities of infection (MOI). After 2 hours of incubation at 37°C, 5% CO2, 1 ml of complete culture medium was added to the cells. Because no visible toxic effect was observed in comparison with the controls (only PBS plus 1 mM MgCl2/1% HS), it was not necessary to remove the virus. Adenoviral transduction of primary lymphoma cells was considered successful if concurrent CD19 expression with green fluorescent protein (GFP) was observed.Transduction of lymphoma cells with CsCl-purified adenovirus was carried out in 24-well plates with 5 × 10The recombinant adenoviral Ad.GFP vector (pQB-AdBM5GFP), an E1- and E3-deleted replication-defective adenovirus type 5 under control of the cytomegalovirus (CMV) promoter, was purchased from Quantum Biotechnologies . The adenovirus vector (Ad.IL-2) containing the human IL-2 sequence was kindly provided by Frank L. Graham, McMaster University, Hamilton, Ontario, Canada . The E1/2 flasks were infected with ~5 plaque-forming units (PFU)/cell in 2 ml of phosphate-buffered saline (PBS) containing 1% horse sera (HS). After 2 hours incubation at 37° in a humidified atmosphere of 5% CO2, the inoculum was replaced by fresh medium (DMEM/2% HS). After 48 h, nearly completely detached 911 cells were harvested and collected in 1 ml PBS/1% HS. Virus was isolated by three cycles of flash-freeze thawing. The lysates were cleared by centrifugation at 3000 rpm for 10 minutes. Viruses were then purified on double cesium chloride gradients and stored in PBS/10% glycerol at -80°C.Production of the adenovirus lots was performed as described previously . Briefly2, the medium was replaced by F-15 minimal essential medium (Life Technologies) containing 1% agarose , 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.4), 0.0025% L-Glutamine, 5% yeast extract, 8.4% NaHCO3, 50 μg/mL streptomycin, 50 μg/mL penicillin, and 2% HS. The titers of the virus stocks were at least 1 × 1010 PFU/ml.Plaque assays were essentially performed as described by Graham and Prevec . BrieflyIL-2 and IL-12 levels in conditioned medium were determined by an enzyme-linked immunosorbent assay (ELISA) method. The ELISA reagents were purchased from Endogen, . Briefly, a microtiter plate was coated with a monoclonal antibody specific for IL-2 or IL-12. The IL-12 antibody recognizes only the p70 heterodimer and neither of the individual subunits, p35 or p40, or the homodimeric form of p40. The cytokines present in samples are bound by the immobilized antibody. After several washes to remove unbound proteins, an enzyme-linked (horseradish peroxidase) polyclonal antibody was added to the wells which binds IL-2 or IL-12. After washing, the substrate solution was added, and the color which developed was measured using a spectrophotometer at a wavelength of 450 nm. The optical density of the samples was then compared to a standard curve.5 PBMC were incubated in 96-well flat-bottom plates  in a final volume of 200 μl per well. After 3 days 20 μl of EZ4U reagent  was added to each well and results were obtained on a multi-well scanning spectrophotometer at 450 nm.An MTT -2,5-diphenyl tetrazolium bromide) based colorimetic assay  was perf51Cr release assay. The procedure is based on loading the target cells with a fluorescence enhancing ligand 2,2:6,2-terpyridine-6,6-dicarboxylate). The hydrophobic ligand penetrates the membrane quickly and within the cell the esterbonds are hydrolysed to form a hydrophilic ligand  which no longer passes the membrane. After cytolysis the ligand is released and introduced to the europium solution. The europium and the ligand form a highly fluorescent and stable chelate (EuTDA). The measured signal correlates directly with the amount of lysed cells.A EuTDA nonradioactive cytotoxicity assay  was used to compare the cytotoxic activity of IL-2 and IL-12 stimulated PBMC against unmodified lymphoma cells . This as6 lymphoma cells were washed and resuspended in 2 ml PBS. 4.5 μl BATDA solution was added and incubated at 37° for 30 min. Then, cells were washed 3 times, resuspended in 100 μl PBS, and incubated in 96-well flat-bottom plates (Nunc) at a density of 10,000 cells/well. 100 μl of effector PBL cells of varying cell concentations were added so that effector to target cell ratio ranged from 5:1 to 20:1. After incubation at 37° for 2 h cells were centrifuged for 5 min at 500 × g and 20 μl of the supernatant was transfered to a new flat-bottom plate. 180 μl of Europium solution was added, and after 15 min incubtion at room temperature the fluorescence was measured in a time-resolved fluorometer . The percent specific release was calculated fromBriefly, 2 × 10Wilcoxon matched-pairs test was used to analyze for statistical significance. A p value < 0.05 was considered significant. Data is presented as the mean ± standard error of the mean (SEM).Lymphoma cell lines, primary lymphoma cells, and CIK cells were transduced with Ad.Flexi-12 at various MOI  and analyzed 72 h later. Transduction efficiencies were determined by GFP expression analysis using a fluorescence-activated cell sorter (FACS). Additionally, cell surface antigens were analyzed by FACS for the expression of CD19, integrin avβ3, integrin avβ5, and CAR. Adenoviral transduction of primary lymphoma cells was considered successful if concurrent CD19 expression with GFP was observed. It was demonstrated that most B cell lymphoma cell lines could be transduced with much higher efficiency than primary tumor samples or CIK cells. At an MOI of 200, up to 40% of Daudi cells and 70% of Raji cells could be transduced Fig. . In cont6 cells and 2.7 ng/ml/106 cells, respectively. In contrast, there was no IL-2 detectable in Ad.IL-2-transduced LAM53 cells. Kinetic analysis of IL-2 production in Raji cells revealed peak secretions between day 2 and 3. IL-2 was detectable until day 8 post-infection  with Ad.GFP as a control vector. Cytokine production was assayed 72 h post-infection. As shown in Fig. tion Fig . Similarlls Fig. . Peak exlls Fig. .To determine if adenoviral-expressed cytokines from transduced lymphoma cells would have an impact on the proliferation rates of PBMC from healthy donors, the following experiment was performed. PBMC were freshly isolated and various concentrations of cytokines (1–1000 pg/ml) either derived from the supernatants of transduced lymphoma cells or recombinant were added. Then, an MTT assay to assess the proliferation rate was performed five days later. For blocking experiments, a neutralizing monoclonal antibody against IL-2 or IL-12 was used. Figure Raji cells were transduced with Ad.IL-2 (MOI 200), Ad.Flexi-12 (MOI 200), or Ad.IL-2 and Ad.Flexi-12 together (MOI 100 each) and co-cultured with PBMC for 72 h. Non-transduced (control) and Ad.GFP transduced Raji cells were used as controls. Stimulated PBMC were harvested and assayed for their cytolytic activity against unmodified lymphoma cells using a EuTDA nonradioactive cytotoxicity assay. It could be shown that Ad.IL-2 transduced lymphoma cells produced a significant p < 0.05) anti-tumor effect but not the combined effect of Ad.IL-2/Flexi-12 or Flexi-12 alone  declare that they have no competing interests.OE and DW designed the experiments and performed the experimental studies presented in this paper. PB developed the experimental protocols and assisted in the analysis of the results. CZ, DF, and ISW participated in the design of the study and its coordination. All authors have read and approved this manuscript.


Abundant nonfibrillar oligomeric intermediates are a common feature of amyloid formation, and these oligomers, rather than the final fibers, have been suggested to be the toxic species in some amyloid diseases. Whether such oligomers are critical intermediates for fiber assembly or form in an alternate, potentially separable pathway, however, remains unclear. Here we study the polymerization of the amyloidogenic yeast prion protein Sup35. Rapid polymerization occurs in the absence of observable intermediates, and both targeted kinetic and direct single-molecule fluorescence measurements indicate that fibers grow by monomer addition. A three-step model  accurately accounts for the distinctive kinetic features of amyloid formation, including weak concentration dependence, acceleration by agitation, and sigmoidal shape of the polymerization time course. Thus, amyloid growth can occur by monomer addition in a reaction distinct from and competitive with formation of potentially toxic oligomeric intermediates. Polymerization of the amyloidogenic yeast prion protein Sup35 occurs by monomer addition in a reaction distinct from formation of potentially toxic oligomeric intermediates Many proteins of diverse sequences, structures, and functions form morphologically similar β-sheet–rich fibrillar aggregates commonly referred to as amyloid . Amyloid+PSI], which results from self-propagating aggregation of the translation termination factor Sup35 and leads to a nonsense suppression phenotype, provides an excellent system for studying amyloid fiber formation and prion propagation . Equilibrium AUC indicated the material was predominantly monomeric, giving fitted average molecular masses of 28.5 ± 1.7 kDa , with no appreciable concentration dependence from 1.5 to 5.8 μM A and 1B.To explore this possibility, we looked at the concentration dependence of the initial rate of growth of soluble NM onto the ends of a well-defined amount of preformed fibers. If a rare oligomer is critical for fiber growth, then mass action dictates that the quantity and rate of formation of such oligomers will be highly concentration dependent. As a consequence, the initial rate of polymerization should depend strongly on the concentration of soluble NM. Using a thioflavin T binding assay that allows continuous measurement of amyloid formation, we measured the rate of amyloid growth after mixing a quantity of soluble NM with a known amount of preformed nuclei. We found that the initial rate of fiber growth was directly proportional to the concentration of soluble NM over a range of 0–1 μM A. This lInterestingly, at higher NM concentrations (>10 μM), the rate of fiber elongation shows a weaker-than-linear dependence on NM concentration. Accumulation of off-pathway aggregates, which we can observe at high [NM], could contribute to such an effect. However, in the presence of moderate levels of denaturant (100 mM guanidine hydrochloride [GuHCl] and 100 mM urea), NM remained monomeric up to at least 20 μM A, and unA model in which growth of NM fibers is largely limited by the encounter rate of monomer and fiber end still has two puzzling features of the polymerization process to explain. First, like many other amyloid formation reactions, NM polymerization is dramatically accelerated by agitation  Figure A, which At a given NM concentration the rate of polymerization is proportional to seed concentration, and therefore the acceleration seen in the agitated reaction implies that the number of fiber ends is increasing with time, perhaps due to the ability of agitation to fragment long fibers. To test this possibility, we prepared long fibers by allowing prolonged growth of NM onto preformed seeds in the absence of agitation, and then subjected the fibers to end-over-end rotation. Prior to rotation, the fibers were very long and had a weak ability to seed polymerization of monomeric NM. After 1 h of rotation, we saw many more fibers of much shorter length, and the seeding activity had increased 10-fold. Further rotation 14 h) produced still shorter fibers and higher seeding activity C and 4D. h produc2) time course  of the length of the lag time on the concentration of NM in unseeded polymerizations . This fie course . We simie course A and 5B.e course . HoweverPrevious work from The advent of single-molecule fluorescence technology enabled an independent and more direct way to test whether fibers grow by monomer addition. Previous work  establisBy following the intensities of single fluorescent addition events, we could directly determine the size of the unit of addition. Oligomer addition (unlike monomer addition) predicts that the fluorescence intensity of a single addition event should depend strongly on the fraction of NM that is fluorescently labeled C. We pre+PSI], form and grow.Amyloid formation is a ubiquitous feature of polypeptides. However, compared to protein folding reactions, for which a wide range of biophysical, structural, and analytical approaches have provided detailed information on the pathways by which native states are obtained, very little is known about the underlying steps by which amyloid fibers assemble and grow. In large part this is due to the complexity of the amyloid formation reaction, which can involve a wide spectrum of on- and off-pathway intermediates. In the present study, we have used a combination of kinetic analysis designed to look at specific mechanistic steps and single-molecule fluorescence to determine how amyloid fibers of Sup35, the prion determinant of the yeast state [+PSI] translation read-through phenotype in vivo. While division of Sup35 prion particles appears to depend on cellular chaperones including Hsp104 (5 M−1 s−1), soluble Sup35 would have a half-life of about 3 min, which is comparable to the time scale of Sup35 translation and much faster than the doubling time of yeast (90 min). Thus, the monomer growth mechanism would lead to the depletion of Sup35 characteristic of the [+PSI] state (see +PSI] and [psi−] are both stable states) and whose aggregated state must be amplified exponentially to keep pace with cell division.A key finding is that Sup35 NM amyloids grow efficiently by the addition of monomers to fiber ends. We also establish that monomer addition, in combination with fiber fragmentation, accurately predicts the otherwise puzzling features of de novo polymerization kinetics. While it is still possible that oligomers could add to the ends of fibers if allowed to accumulate, we find that monomer addition is rapid and efficient. This monomer addition mechanism can account for the generation of the -N-piperazinylamido}hexyl D-biotinamide, hydrochloride) was purchased from Dojindo . Alkali-soluble casein (5%) was purchased from Novagen .Cy3 mono maleimide Gold and Cy5 mono maleimide Gold were purchased from Amersham . Alexa Fluor 647 CSup35 residues 1 to 254 (NM) C-terminally tagged with 7×-histidine were purified as reported previously .2 maleimide  in 25 mM sodium phosphate buffer containing 6 M GuHCl and 0.1 mM TCEP (pH 8.0) at 4 °C overnight with 67% efficiency. Efficiency of labeling was determined from absorbance at 275 nm and at the excitation maximum of the dyes, correcting for absorbance of the dye at 275 nm. Biotinylated NM was made analogously using biotin-PEAC5-maleimide  with 1 mM TCEP, obtaining about 20% efficiency.NM with a single cysteine inserted after the polyhistidine tag was labeled by Cy3 mono maleimide Gold, Cy5 mono maleimide Gold, or Alexa Fluor 647 Chttp://www.analyticalultracentrifugation.com.Both velocity and equilibrium AUC were performed in a Beckman Optima XL-A analytical ultracentrifuge using an An60Ti-Rotor at 20 °C. Protein was in buffer C (5 mM potassium phosphate and 150 mM sodium chloride [pH 7.4]). Velocity sedimentation was analyzed at a speed of 40,000 rpm at NM concentrations of 3.7, 6.2, and 12.9 μM (determined by absorbance at 275 nm). Data were collected with three replicates at radial steps of 0.003 cm and scans every 9 min. Data were analyzed with Sedfit using the c(s) method . EquilibFor continuous thioflavin T assay measurements see B, and 5DDiscrete thioflavin T assay measurements see E, and 4BFor polymerizations followed by Alexa Fluor 647 fluorescence see C and 2F,Seeds used were produced by polymerization of 2.5 μM NM followed by sonication with a Fisher Scientific Sonic Dismembrator (Model 500) fitted with a microtip for 60 s , new growth of unlabeled NM off of preformed fibers made from NM tagged with an HA epitope was measured as previously described . The HA Numerical integration was performed using Euler's method implemented in a C program (code available upon request). The following is a general description of the strategy used for numerical modeling. A set of differential equations was written to account for the following steps: formation of a new stable nucleus from a defined number (n) of monomers, growth of fibers by monomer addition (limited by the rate of encounter of monomer and fiber end), and production of new fibers by fragmentation of existing fibers. Fiber growth was modeled to be irreversible  , becauset = time; x = concentration of monomers; yi = concentration of fibers containing i monomers; y = concentration of all fibers of length n or greater; n = the number of monomers in the smallest stable species; BreakDep = the power to which the rate of fragmentation of a fiber depends on its length. For all simulations this value was set to 3, based on theoretical estimates for polymers  term accounts for fragmentation of all fibers long enough to break and give a resulting fiber of length i. The sum begins at j = (i + n + 1) because no smaller fiber could break to give a fragment of size i, leaving another fragment of at least size (n + 1).Here, j represents fiber length (the number of monomers in a fiber) and the sum is taken over all values of j greater than or equal to (i + n + 1). The nuc = effective fiber nucleation rate constant; kgrowth = fiber elongation rate constant; kbreak = fiber fragmentation rate constant. The equations are as follows.Differential equations for modeling contain the following terms: kFor monomer concentration:nuc*n*xn term accounts for the loss of n monomers during the formation of each stable nucleus. Stable nuclei are modeled to form at a rate of knuc*xn. The kgrowth*x*y term accounts for the loss of one monomer for each fiber elongation event; each fiber grows at a rate of kgrowth*x and there are y total fibers.The kFor the smallest stable species:nuc*xn term accounts for the spontaneous formation of new nuclei. The kgrowth*x*yn term accounts for the disappearance of fibers of length n as they grow into longer fibers.The kFor short fibers :growth*x*yi−1 and lost by growth into longer fibers at a rate of kgrowth*x*yi, accounting for the first term. The second term accounts for formation of fibers of length i by fragmentation of longer fibers (see description of zabove(i) above). There is no loss of fibers of these lengths from fragmentation because they are assumed to be too short to break.Fibers of length i are created by elongation of shorter fibers at a rate of kFor fibers longer than twice the size of the nucleus plus one monomer:break is a rate constant for the fragmentation process, yi is the number of fibers of this length, (i − 2*n − 1) is the number of points along the length of the fiber where it could break, and iBreakDep accounts for the tendency of longer fibers to break more easily than shorter fibers (see the description of BreakDep above). There are (i − 2*n − 1) places the fiber could break because there are (i − 1) monomer-monomer interfaces in a fiber of length i and breaking at any of the n interfaces closest to either end of the fiber would give one fiber of size n or smaller.The first two terms are the same as for the short fibers. The additional term accounts for fragmentation of fibers of length i into shorter fibers. This term is arrived at in the following way: ky = A(cosh (Bt) − 1), where t is time and A and B are parameters to be fit with the restriction that AB2 scales as [NM]n+1 .Curve fitting of experimental data to the linearized model of z = total amount of NM in amyloid, and zfinal = total amount of NM in amyloid at the end of the reaction, then relative fiber number = (dz/dt)/. Values for z were measured by thioflavin T fluorescence (continuous thioflavin T assay). The equation used for relative fiber number comes from the observation (see z/dt) is proportional to both fiber concentration (relative fiber number) and soluble NM concentration . The relative fiber number versus time was fit to an exponential (y = Aetb) by nonlinear least squares regression as described above , and controlled by the QED in vivo software package . Images were acquired digitally with a Mega-10 intensified CCD camera  and analyzed using ImageJ  and MATLAB software. Samples were analyzed in glass-bottom microwell dishes  which were prepared by application of 60 μl of biotinylated BSA (1 mg/ml) for 20 min followed by washing with buffer, application of 60 μl of streptavidin (0.2 mg/ml) for 20 min, washing, application of 100 μl of casein (5%) for 30 min to 2 h, washing, application of 40 μl of Cy5-labeled fibers for 10 min, and finally washing with buffer. The fibers were prepared in a 300-μl reaction with 1% (v/v) seed (sonicated from a 2.5 μM NM reaction). The sonicated seed was grown for 15 min with NM at 2.5 μM . The seed was diluted 2-fold and sheared with a 200-μl pipet tip before application to the slides. The slides were kept in buffer until use. Buffer was removed and Cy3-labeled NM  was added to the slide immediately before viewing. For addition studies, a Cy5 image was taken to locate the fiber ends and then images were taken every 15 s in the Cy3 channel to look for events at fiber ends.AFM analysis established that NM fibers grow at an average rate of 100 nm/min at a soluble NM concentration of 2.5 μM and can grow at least a factor of two faster at higher concentrations. Estimating one monomer per 3 nm of length in a fiber (from the approximate monomer volume and a fiber diameter of 4.5 nm from AFM) gives a rate of at least one per second. This is a conservative estimate  because larger fiber diameters have been measured by electron microscopy , indicat5 M−1 s−1, and estimating 200 seeds .Click here for additional data file.The GenBank accession number for Sup35p is NP_010457.


In recruiting several of these regulatory factors to the 5' long terminal repeat (LTR) promoter during transcription elongation, HIV-1 modulates replication of its genome in a process mediated by the virus-encoded transactivator Tat. One particular cellular regulatory factor, DSIF subunit human SPT5 (hSpt5), has been implicated in both positively and negatively regulating transcriptional elongation but its role in Tat transactivation in vivo function of hSpt5 and define its role in Tat transactivation and HIV-1 replication, we used RNA interference (RNAi) to specifically knockdown hSpt5 expression by degrading hSpt5 mRNA. Short-interfering RNA (siRNA) designed to target hSpt5 for RNAi successfully resulted in knockdown of both hSpt5 mRNA and protein levels, and did not significantly affect cell viability. In contrast to hSpt5 knockdown, siRNA-mediated silencing of human mRNA capping enzyme, a functionally important hSpt5-interacting cellular protein, was lethal and showed a significant increase in cell death over the course of the knockdown experiment. In addition, hSpt5 knockdown led to significant decreases in Tat transactivation and inhibited HIV-1 replication, indicating that hSpt5 was required for mediating Tat transactivation and HIV-1 replication.To understand the bona fide positive regulator of Tat transactivation and HIV-1 replication in vivo. These results also suggest that hSpt5 function in transcription regulation and mRNA capping is essential for a subset of cellular and viral genes and may not be required for global gene expression.The findings presented here showed that hSpt5 is a  Tat,nscripts . Cellulanscripts -14. Compnscripts -18. Tat nscripts ,14,17,19nscripts .in vitro to phosphorylate both hSpt5 and CTD [Human DSIF consists of subunits hSpt5 and hSPT4 and was originally discovered as a negative elongation factor that binds to RNA pol II . In conj and CTD ,10,21-29 and CTD -32. Curr and CTD .in vitro studies have shown that hSpt5 is required for Tat transactivation and that both hSpt5 and RNA pol II phosphorylation is stimulated after recruitment of P-TEFb by Tat [in vivo remains unclear.Although hSpt5's role in transcription regulation in association with P-TEFb has been established, its involvement in Tat transactivation and HIV-1 replication continues to be elucidated. Several b by Tat ,29,34. hb by Tat ), since b by Tat -43. Howeb by Tat . Therefoin vivo and further defined hSpt5 cellular functions. RNAi is a remarkably efficient process whereby double-stranded RNA (dsRNA) induces sequence-specific degradation of homologous mRNA in animal and plant cells  to address whether hSpt5 is required for Tat transactivation and thus HIV-1 replication  in ref. ). In mam in ref. ,46. RNAi in ref. -52. Thes in ref. ,52-54 an in ref. , which h in ref. . RNAi ha in ref. ,57). In To inhibit hSpt5 expression in a cultured human cell line using RNAi, siRNA targeting an hSpt5 sequence from position 407 to 427 relative to the start codon was designed Figure . Magi ceHaving established that hSpt5 could be knocked down using RNAi, the kinetics of hSpt5 knockdown were examined. To perform kinetic experiments, hSpt5 duplex siRNA, single-stranded antisense hSpt5 siRNA, or mismatch duplex hSpt5 siRNA were transfected into Magi cells. Cell lysates were collected at various time points to assay for protein levels during hSpt5 knockdown. Immunoblot analysis using anti-hSpt5 antibodies revealed the timing of gene suppression and persistence of hSpt5 RNAi effects in Magi cells during the time course experiment Figure . hSpt5 kKnowing that the kinetics of hSpt5 peaked at 42–54 h post-transfection, we were able to evaluate the viability of cells during hSpt5 knockdown experiments over varied time intervals. Cell viability was assessed using trypan blue exclusion at various times after a single transfection of various siRNAs. As shown in Figure C. elegans [hSpt5 has been shown to interact with the human mRNA capping enzyme (HCE) and this interaction enhances capping enzyme guanylylation and mRNA capping several fold . Since S elegans . These rin vivo, Tat transactivation during hSpt5 knockdown in Magi cells was monitored. Magi cells are a HeLa cell line harboring a stably integrated single copy of the HIV-1 5' LTR-β-galactosidase gene. These cells are also genetically programmed to express the CD4 receptor for HIV-1 infection  and mock-treated cells not transfected with pTat-RFP was determined. The results of this quantitation are shown in Figure NL-GFP, an infectious molecular clone of HIV-1. Knockdown of hSpt5 protein levels was then evaluated 48 h post infection in doubly transfected cells. An even larger decrease in hSpt5 protein levels was observed in doubly transfected cells (~95% knockdown) as compared to singly transfected cells -methyl ether-protected oligos . Synthetic RNAs were deprotected, annealed and purified using standard protocols provided by the manufacturer. Formation of duplex RNA was confirmed by 20% non-denaturing polyacrylamide gel electrophoresis (PAGE). Sequences of siRNA duplexes were designed as described previously  and subjmultinucleate activation of galactosidase indicator) cells carrying the endogenous HIV-5'LTR β-galactosidase gene were maintained at 37°C in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum (FBS), 0.2 mg/ml of Geneticin (G418) and 0.1 mg/ml Hygromycin B . Cells were passaged at sub-confluence and plated at 70% confluency for transfection. Reporter plasmids and siRNA were co-transfected into Magi cells using Lipofectamine (Invitrogen) in duplicate 6-well plates . A transfection mixture containing 25–150 nM siRNA and 9 μl of Lipofectamine in 1 ml of serum-reduced OPTI-MEM (Invitrogen) was added to each well. For high efficiency knockdown experiments, 150 nM siRNA was used. After incubating at 37°C for 6 h, cells were cultured in antibiotic-free DMEM. For further analysis, transfected cells were harvested at various time intervals, washed twice with phosphate buffered saline , flash frozen in liquid nitrogen, and stored at -80°C.Magi (Taq (Invitrogen) and 40 cycles of amplification. Each RT-PCR reaction included 100 ng total cellular mRNA, gene-specific primer sets for hSpt5 and hCycT1 amplification (0.5 μM for each primer), 200 μM dNTP, 1.2 mM MgSO4 and 1U of RT/platinum Taq mix. Primer sets for hSpt5 produced 2.6 kb products while hCycT1 produced 1.8 kb products. RT-PCR products were resolved on a 1% agarose gel and viewed by ethidium bromide staining. Forward and reverse primer sequence for amplifying SPT5 were 5'-ATGTCGGACAGCGAGGACAGC-3' (nts 1–21) and 5'-TGTACATGGCCGGCGTCCC-3' (nts 2638–2656), respectively. Forward and reverse primer sequences for amplifying hCycT1 are 5'-GCAACAAGTTCAAGATCTGGTCAT-3' (nts 381–404) and 5'-CCCGGGGGATCCTTACTTAGGAAGGGGTGGAAGTGG-3' (nts 2158–2200); underlined sequences represent restriction enzyme sites), respectively.Total cellular mRNA was prepared from HeLa cells transfected without siRNA or with hSpt5 or control siRNAs using a Qiagen RNA mini kit, followed by an oligotex mRNA mini kit (Qiagen). RT-PCR was performed using a SuperScript One-Step RT-PCR kit with platinum siRNA treated cells were harvested as described above and lysed in 1X reporter lysis buffer (Promega). After centrifugation to remove cellular debris, concentrations of proteins in lysates were determined using a Dc protein assay kit (Bio-Rad). Proteins in 30 μg of total cell lysates were fractionated by 10% SDS-PAGE, transferred onto a polyvinylidene difluoride membrane , and immunoblotted with antibodies against hSpt5 (Pharmingen) and hCycT1 (Santa Cruz Biotech). Protein content was visualized by a BM chemiluminescence Blotting Kit . The blots were exposed to X-ray film (Kodak MR-1) for various times (between 1 s and 5 min).Magi cells were harvested 48 h after transfection with Tat-RFP plasmids in the absence or presence of siRNAs. Cell lysates were prepared and quantified as described above. To perform standard β-galactosidase assays, 120 μg of cell lysates were mixed in 150 μl of 1X reporter lysis buffer and 150 μl of 2X β-galactosidase assay buffer (Promega), and incubated at 37°C for 30 min. To stop the reaction, 500 μl of 1 M sodium carbonate was added to the mixture and mixed well by vortexing briefly. Absorbance of the reaction mixture was read immediately at 420 nm. The amount of Tat-RFP protein was determined using a fluorescence spectrophotometer . 300 μg of cell lysates was subjected to the spectrophotometer with slit widths set at 4 nm for both excitation and emission wavelengths as described previously ,64. Fluo5 cells per well) and transfected with siRNAs as previously described [4 cells per well), incubated 3 h and infected with HIVNL-GFP, an infectious molecular clone of HIV-1. HIV-1 virions  were added in doubling dilutions to duplicate wells. 48 h post infection, cells were harvested to quantify β-galactosidase activity and protein levels.HeLa-CD4-LTR/β-gal indicator (Magi) cells  were plaescribed . siRNA (The author(s) declare that they have no competing interests.Y-HP carried out all the Spt5 silencing experiments, C-yC performed capping enzyme knockdown experiments, HC performed quantitative analysis of the data, J-MJ performed HIV-1 replication assays, and MS analyzed and interpreted HIV inhibition results. TMR conceived the ideas, and participated in the experimental design and in drafting the manuscript.


Renal artery stenosis (RAS) is a known cause of hypertension and ischemic nephropathy. Stenting of the artery is a valid approach, in spite of cases of unexpected adverse evolution of renal function.In this study, 27 patients with unilateral RAS were subjected to stenting and followed for a period of one year, while 19 patients were observed while on medical treatment only. The group of 27 patients, 67.33 ± 6.8 years of age, creatinine of 2.15 ± 0.9 mg/dl, following stenting, were followed at intervals with biochemical tests, renal scintigraphy and doppler ultrasonography. The control group  was also followed for one year.One year after stenting mean creatinine clearance (Ccr) increased from 36.07 ± 17.2 to 40.4 ± 21.6 ml/min (NS). Arterial BP, decreased after 1,3,6, and 12 months (p < 0.05). The number of antihypertensive drugs also decreased (p < 0.05). A significant increase in proteinuria was also observed. In the control group both Ccr, BP and proteinuria did not show significant changes. Based on renal scintigraphy and Ccr at subsequent times, it was possibile to evaluate the timecourse of renal function in both kidneys of the stented patients. In the stented kidneys Ccr increased significantly. On the controlateral kidney a decrease of renal function (p < 0.05) was observed. Resistance index appeared to be a risk factor of the functional outcome.Stenting of RAS due to atherosclerosis is followed by stabilization or improvement of Ccr, mainly at the stented kidney, while contralateral renal function showed a decrease. Renal artery stenosis due to atherosclerotic changes of the renal arteries has become a serious concern as a cause of hypertension and renal ischemia, resulting frequently in end-stage renal failure ,2. SeverThe study has been carried out prospectively on 46 patients affected by hemodynamically significant atherosclerotic renal artery stenosis, detected by Magnetic Resonance Angiography or Selective Digital Angiography. All the patients had a unilateral stenosis. 27 patients (diabetes mellitus in 8 cases) were subjected to stenting of the stenotic renal artery while 19 patients (diabetes mellitus in 9 cases) were kept on medical treatment only. Clinical data of the two groups are reported in table In all these patients the renal artery was approached through the femoral artery. French 6 guiding catheter (type "Cobra"or "Bates") was used for selective renal artery angiography and for positioning the stent. All stenotic lesions were repaired with stainless stent Express Vascular SD Monorail 5.5-6-15/20 mm.premounted on a balloon catheter on Choice extra support 014" guide. In these cases primary stenting was performed. The procedure requires, usually, an injection containing 30 ml of 50–50 mixture of isotonic contrast and normal saline.The patients were followed at the outpatient clinic of the Nephrology unit. Duplex-doppler sonography and renal scintigraphy were carried out basally and following 1,3,6 and 12 months after the stenting procedure. At the same times, biochemical parameters, like serum creatinine and proteinuria were measured. Creatinine clearance was evaluated with the Cockcroft and Gault formula .Doppler ultrasonography was carried out after fasting, following a three days of a low fibre diet and without smoking for a minimum of six hours before the procedure. Patients were studied with an Acuson 120 XP/4 , equipped with a 3.5 MHz transducer, with longitudinal anterior, lateral and oblique approach, with at least threefold sampling of parameters along the artery. The standard criteria for the diagnosis of significant renal artery stenosis have been previously reported [Renal radionuclide scintigraphy was performed with a gamma camera  with 99mTc-DTPA or with 99mTc-MAG3 (mercaptoacetyltriglycine). MAG3 was chosen in patients with creatinine clearance <25 ml/min. Diuretics and/or ACE inhibitors were discontinued at least three days before, if treatment was underway. The criteria of positivity have previously been reported [Evaluation of single kidney renal creatinine clearance was performed by measurement of creatinine clearance and simultaneous renal scintigraphy with MAG3, with percentage-wise function of each kidney, enabling to calculate the creatinine clearance pertaining to each kidney. The entire set of data for this evaluation was available in 21/27 patients.Statistical analysis. A descriptive univariate analysis, consisting in evaluation of percentages, means and standard deviations has been carried out as first step. To evaluate the dependence among the nominal variables, a Parson's Chi square test was also carried out. In case of not applicable Chi square test due to low theoretical frequencies (<5), Fischer exact test for tables 2 × 2 was employed. Comparison of means of the two groups was made. Since the data did not show a normal distribution, non parametric tests, as Wilcoxon rank-sum test for paired data and Mann-Whitney test, were employed. The significance level was <0.05, as usual. The more interesting significant and not significant data were represented graphically. The data were evaluated with the statistical package BMDP Release 7 .Clinical and biochemical data of the treated and control groups are reported in Table During 12 months observation period one patient of the stented group began dialysis treatment, while in the control group 4 patients died of cardiovascular events and one patient started the dialysis treatment.A significant drop in systolic and diastolic blood pressure at all control times compared to basal values, was found in the stented patients, while no significant blood pressure drop was found in the patients not undergoing the PTA stenting procedure Table . A signiA significant increase in proteinuria following the stenting was found at 1 and at 12 months, while the increment in proteinuria observed at the other control times was only borderline significant. There was no diference in the increase of proteinuria between patients with and without diabetes mellitus. No changes in proteinuria was observed in the control group.An increase in creatinine clearance and a slight fall in serum creatinine, however not reaching a significance level, was observed in the stented group, while no increment in creatinine clearance was found in the control group Table . This obIn addition, patients with the stent were divided in those cases with a RI above 0.80 and cases with this parameter below 0.80. The patients with lower RI improved, on average, renal function while the patients with elevated RI had a worse outcome Fig. . RI valuThe advantage deriving from positioning a stent in a significantly stenotic renal artery has been debated in recent years. Generally favorable results have been reported by Dorros et al  on a widIn our experience, the fall in blood pressure and of the number of antihypertensive drugs was confirmed. Our results on the overall outcome of renal function, over a one year observation period, in patients with one sided renal artery stenosis of atherosclerotic origin, have been satisfactory. As a risk factor of worse outcome, our data have confirmed that RI above 0.80, results in a less satisfactory outcome compared to patients with RI lower than 0.80, as already reported by Radermacher et al. . The steAs for the increment in proteinuria observed following stenting of renal artery, this finding has not been reported previously, while in basal conditions significant proteinuria in patients with renal artery stenosis has been already reported in the literature . TherefoLess is known of the renal function in the kidney affected by the arterial stenosis, following the stenting procedure, compared to the contralateral kidney. In all our patients renal artery stenosis was of atherosclerotic origin, without cases of fibromuscular dysplasia. In general, no adverse events were found following the stenting in the patients closely followed for one year. In our experience, there were no apparent cases of cholesterol embolization, of thrombosis of the artery, of occlusion of the stent, or of dissecation of the renal artery. The function of the stented kidney improved in most of patients while a reduction of renal function was observed in the controlateral kidney. The overall renal function was stable. Similar findings have been published by Airoldi et al., Leertouwer et al., and La Batide-Alanore et al. ,13,14. HThe reduction in contralateral kidney function has been attributed to ultrafiltration in the non stenotic kidney, declining after stenting of the stenotic contralateral renal artery. In addition, hemodynamic factors consequent to a decrease in renin-angiotensin activity could be considered as a factor. The involvement of the renin angiotensin system in renal artery stenosis should be suspected due to the significant fall in systolic and diastolic blood pressure following the procedure, an occurrence not found in the control group. Actually, a fall in plasma renin activity or concentration following dilatation of the stenotic artery has been reported by Airoldi et al  and by LIn conclusion, the stenting procedure of a stenotic renal artery does not seem to carry important risks, and is accompanied by a definite improvement of the stented kidney, with some reduction of the filtration rate of the controlateral kidney. This event cannot be considered unfavorable, since it denounces a condition of hyperfiltration of the kidney, probably, if left unchanged, able to induce a deterioration of renal function with time. Therefore, also in case of overall stabilization of renal function following the stenting procedure, improvement of the stented side and reduction of hyperfiltration on the contralateral side are both favorable evolutions for long-term success of the revascularization procedure. The results are less satisfactory in patients with RI >0.80. They should probably be excluded from the stenting procedure.The author(s) declare that they have no competing interests.GC, conceived the protocol and cohordinated the studyEM, participated in the design of the study and in the cohordinationCC, was encharged of the magnetic resonance image analysisRL, collaborated in the duplex doppler ultrasonography examinationIN, was responsible of the statistical examination of dataGR, responsible of renal scintigraphic investigationDS, performed all the biochemical testsAZ, performed arteriography and stenting of renal arteriesRC, was active in the ultrasonography investigation and screening of patients with renal artery stenosisThe pre-publication history for this paper can be accessed here:


The non-invasive nature of laser biostimulation has made lasers an attractive alternative in Medical Acupuncture at the last 25 years. However, there is still an uncertainty as to whether they work or their effect is just placebo. Although a plethora of scientific papers published about the topic showing positive clinical results, there is still a lack of objective scientific proofs about the biostimulation effect of lasers in Medical Acupuncture. The objective of this work was to design and build a low cost portable laser device for stimulation of acupuncture points, considered here as small localized biosources (SLB), without stimulating any sensory nerves via shock or heat and to find out a suitable method for objectively evaluating its stimulating effect. The design is aimed for studying SLB potentials provoked by laser stimulus, in search for objective proofs of the biostimulation effect of lasers used in Medical Acupuncture.The proposed biostimulator features two operational modes: program mode and stimulation mode and two output polarization modes: linearly and circularly polarized laser emission. In program mode, different user-defined stimulation protocols can be created and memorized. The laser output can be either continuous or pulse modulated. Each stimulation session consists of a pre-defined number of successive continuous or square pulse modulated sequences of laser emission. The variable parameters of the laser output are: average output power, pulse width, pulse period, and continuous or pulsed sequence duration and repetition period. In stimulation mode the stimulus is automatically applied according to the pre-programmed protocol. The laser source is 30 mW AlGaInP laser diode with an emission wavelength of 685 nm, driven by a highly integrated driver. The optical system designed for beam collimation and polarization change uses single collimating lens with large numerical aperture, linear polarizer and a quarter-wave retardation plate. The proposed method for testing the device efficiency employs a biofeedback from the subject by recording the biopotentials evoked by the laser stimulus at related distant SLB sites. Therefore measuring of SLB biopotentials caused by the stimulus would indicate that a biopotential has been evoked at the irradiated site and has propagated to the measurement sites, rather than being caused by local changes of the electrical skin conductivity.A prototype device was built according to the proposed design using relatively inexpensive and commercially available components. The laser output can be pulse modulated from 0.1 to 1000 Hz with a duty factor from 10 to 90 %. The average output power density can be adjusted in the range 24 – 480 mW/cm2, where the total irradiation is limited to 2 Joule per stimulation session. The device is controlled by an 8-bit RISC Flash microcontroller with internal RAM and EEPROM memory, which allows for a wide range of different stimulation protocols to be implemented and memorized. The integrated laser diode driver with its onboard light power control loop provides safe and consistent laser modulation. The prototype was tested on the right Tri-Heater (TH) acupuncture meridian according to the proposed method. Laser evoked potentials were recorded from most of the easily accessible SLB along the meridian under study. They appear like periodical spikes with a repetition rate from 0.05 to 10 Hz and amplitude range 0.1 – 1 mV.The prototype's specifications were found to be better or comparable to those of other existing devices. It features low component count, small size and low power consumption. Because of the low power levels used the possibility of sensory nerve stimulation via the phenomenon of shock or heat is excluded. Thus senseless optical stimulation is achieved. The optical system presented offers simple and cost effective way for beam collimation and polarization change. The novel method proposed for testing the device efficiency allows for objectively recording of SLB potentials evoked by laser stimulus. Based on the biopotential records obtained with this method, a scientifically based conclusion can be drawn about the effectiveness of the commercially available devices for low-level laser therapy used in Medical Acupuncture. The prototype tests showed that with the biostimulator presented, SLB could be effectively stimulated at low power levels. However more studies are needed to derive a general conclusion about the SLB biostimulation mechanism of lasers and their most effective power and optical settings. Nowadays lasers are widely used in therapy and diagnostics. They have been adapted to many medical procedures ranging from surgery, oncology, physiotherapy, dentistry, dermatology and biostimulation. The non-invasive nature of laser biostimulation have made lasers an attractive alternative in Medical Acupuncture at the last 25 years. Unfortunately, there is still an uncertainty as to whether they work or their effect is just placebo. Although a plethora of scientific papers published about the topic showing positive clinical results, there is still a lack of objective scientific proofs about the biostimulation effect of lasers in Medical Acupuncture.The properties of acupuncture points, considered here as small localized biosources (SLB), have been extensively studied over the past 50 years. Research has shown SLB to be small area body regions, which exhibit unique, electrical, physiological and anatomical properties . They are considered to form groups, each group being arranged along a line, called meridian and related to an internal organ -4. SLBs The objective of this work was to design and build a low cost portable laser device for effectively stimulation of SLB without exciting sensory nerves, and to find out a suitable method for objectively evaluating its efficiency. The attempt to define the optimal device parameters was based on the SLB properties, data about existing devices for low level laser therapy and on preliminary measurements performed in our laboratory. The latter suggest that the effect of SLB stimulation is also dependent on the polarization of the coherent emission in addition to its intensity, wavelength and modulation frequency. Therefore the device should provide a polarization adjustment, wide range of modulation frequencies, precise power settings and to have minimum size and cost.The block diagram of the proposed biostimulator design is shown in Fig. The variable parameters of the laser output are: average output power, pulse width, pulse period, and sequence duration and repetition period. After each input parameter is selected, the total energy that would be delivered at the end of the stimulation session is automatically calculated and displayed. When defining the stimulation protocol, the software program reads the selected parameter value and automatically re-calculates the possible set of the other parameters, so that the user could not select inconsistent values or ones that would result in a total energy delivered that exceeds a certain safety limit. In stimulation mode the laser stimulus is applied according to the pre-programmed protocol.θ between the electric field vector of the incident linearly polarized beam and the quarter-wave retarder principal plane is +45 degrees, the emergent beam is circularly polarized as shown in Fig. θ to -45 degrees reverses the sense of the circular polarization. The output from a single cavity laser diode is mainly linearly polarized, parallel to the laser junction. Although, spontaneous emission with a random polarization and with a polarization perpendicular to the laser junction is also present. For a diode operating near its maximum power the polarization ratio is typically greater than 100:1 but when operating near the threshold point, the ratio is considerably lower as spontaneous emission becomes more significant. Therefore a collimating lens followed by a linear polarizer is used since the retarder requires linearly polarized and normally incident light upon its plate over the whole power range. A single collimating lens, with good anti-reflection coating and large numerical aperture to efficiently capture the widely divergent perpendicular axis of the laser diode, is the most efficient and cost effective solution for the current application. Polarizers typically utilize birefringence, dichroism, optical activity, and polarization by reflection or by a metallic thin film [A quarter-wave retardation plate realizes the laser output polarization change, as shown in Fig. The application can tolerate an elliptical beam shape and waveform aberrations, so circularization of the laser beam or correction of the waveform aberrations is not required.The best way for testing the device efficiency is to obtain a biofeedback from the site of stimulation. A suitable non-invasive method is to measure the surface biopotential of the irradiated SLB site. However simultaneous stimulation and biopotential recording from a single SLB is technically difficult and inadequate, since the record may contain sham potentials due to local changes of the electrical conductivity of the irradiated skin. So a new method is proposed to avoid this problem. The method uses separate stimulation and measurement sites. Thus the laser stimulus is applied at SLB situated at the beginning of the meridian (e.g. the first point), where biopotential records are obtained from all the other easily accessible SLB lying distantly on the same meridian see Fig. . TherefoThe schematic of the practical biostimulator circuit, built according to the proposed design is shown in Fig. • RA0-RA2 – outputs used as LCD control signals• RA3-RA4 – outputs, used as DAC control signals• RB0-RB3 – either inputs or outputs, shared between the input control buttons B1-B4 and the LCD data bus DB4-DB7• RB4-RB6 – outputs, used as control signals for the laser diode driverThe LCD is implemented with the dot matrix alphanumeric character module U4 (Seiko Instruments L1682). It features low power consumption, high contrast, wide viewing angle, on-board controller and LSI driver (Samsung S6A0069). All functions required for the LCD drive are internally provided on the chip. Its internal operation is determined by signals sent from the microcontroller. These signals include:• Register select – RS• Read/Write – R/W• Data bus – DB4-DB7 (configured as inputs)• Read/Write Enable – EWhen ports RB0-RB3 are configured as inputs, the LCD data inputs DB4-DB7 have no practical influence on the logic levels set by push buttons B1-B4. LCD operation is also not affected since DB4-DB7 content is read only on logic high at E (U4-pin 6), set by the microcontroller . When pobias and the active power levels of the laser are set [bias level input (U1-pin 14) and at the active level input (U1-pin 3). These inputs drive track and hold amplifiers with hold capacitors C4 and C5. The input voltage range on both inputs ranges from Vref to Vref + 1.6 V, requiring an offset of Vref to be created for common based signals. The bias level is chosen to be equal to Vref, where the active level is determined by the circuit realized with op-amp U5. It performs the level shift and scales the DAC output from Vref to Vref + 1.6 V. This solution is attractive because both DAC and op-amp can run off a single 5 V supply, and the op-amp does not have to swing rail-to-rail. The op-amp U5 output voltage level is given by:The laser diode driver is implemented with the highly integrated circuit U1 , which combines a very fast output current switch with onboard analog light power control loops. It gets feedback current from the laser diode built-in photo detector (U1-pin 8), feeds it to a transimpedance amplifier (TZA) and then to two analog feedback loops where the  are set . The twobias control loops is periodically calibrated via U1-pin 15, where the active control loop is continuously calibrated via U1-pin 1. Resistors R3 and R4 are used to avoid floating of inputs U1-pin 15 and U1-pin 2 when microcontroller ports are in a high impedance mode. The laser pulse modulation is done by switching between the bias and the active power levels according to the logic level at U1-pin 2, where logic high turns the modulation current on. The gain resistor R5 matches the feedback loop transfer function to the laser/photo diode D1. Capacitor C6 optimizes the TZA response, with larger values to slow TZA response. Lower values increase TZA bandwidth but may cause oscillations. When input U1-pin 16 is logic high, the onboard disable circuit turns off the output drivers and returns the light power control loops to a safe state. It is used during initial power up of U1 and when the laser is inactive. In case that input U1-pin 16 floats (after POR or other reset conditions) the driver is disabled. When U1 is re-enabled the control loops are recalibrated.Since the monitor current is proportional to the laser diode light power, the feedback loops effectively control the laser power to a level proportional to the analog inputs. The dd as reference (U3-pin 8) filtered by R12 and C9. The DAC's full-scale output voltage ranges from 0 to Vdd. Special attention was paid to the PCB layout design to minimize the crosstalk between analog inputs and digital outputs.The DAC is implemented with the single 8-bit voltage output MAX517 (U3). It is controlled by the microcontroller via 2-wire serial interface , operates from a single power supply and swings rail-to-rail. POR ensures the DAC output is at zero volts when power is initially applied. It uses the power supply V2), optimized for 670 nm. The polarizer used is a linear polarizing film produced by aligning long chain polymers, which is then laminated in cellulose acetate butyrate (CAB) for durability and stiffness [The drawing of the practical optical system assembly is shown in Fig. tiffness . The quaA prototype device was built according to the proposed design using inexpensive and commercially available components. Its optical and electrical characteristics are given in Table The biostimulator was tested on the right Tri-Heater (TH) meridian of ten subjects according to the proposed method see Fig. . The lasThe noise present in the signals is mainly composed of electromyographic signals and noise from the electrode-skin interface. The first two signal records (TH-8 and control) contain ECG artifacts and additional electromyographic noise since those two electrodes were positioned relatively distant from the reference electrode. The frequency bandwidth was limited to 200 Hz by sixth order low-pass Bessel filter and the signals were sampled with 1 kHz.Recording to the EU regulation  this device falls under class IIb in order to obtain the CE mark. The biostimulator prototype was categorized as Class IIIb laser product according to the International Standard for the Safety of Medical Laser Products IEC 601-2-22, as stated in Table The best solution for building a compact hand held biostimulator would be to design a custom made integrated circuit, but the cost would be much higher. We found a good alternative in using surface mount technology (SMT), commercially available integrated laser diode driver and a RISC Flash microcontroller. This solution resulted in a reduction in parts, size and power consumption. The proposed method for testing the device efficiency is very sensitive to precise electrode and stimulus positioning. Even a deviation of 3 mm from the exact SLB location may prevent the recording electrode from capturing signals from the source. The same deviation of the stimulus position also results of ineffective excitation of the targeted SLB and thus no SLB evoked potentials can be recorded. The method is also susceptible to the electrode-skin pressure, but not only due to its strong influence on the contact impedance. It was observed that the excessive electrode-skin pressure led to diminishing or even disappearing of the SLB signal, although the contact impedance was lower. This is most probably due to the pressure exerted on the SLB source that may affect the signal generation or transduction. Alternatively insufficient electrode-skin pressure led to excessive contact impedance and noise from the electrode-skin interface. The preliminary results suggest that a circularly polarized laser emission is most effective when used on the so-called Yang acupuncture meridians but not on Yin types. However more studies are needed to validate or disprove this observation.The specifications of the prototype, built according to the proposed design, were found to be better or comparable to those of other existing devices. It features small size and low component count and power consumption. Because of the low power levels used the possibility of sensory nerve stimulation via the phenomenon of shock or heat is excluded. Thus senseless optical stimulation is achieved. The optical system presented offers simple and cost effective way for beam collimation and polarization change. The novel method proposed for testing the device efficiency allows for objectively recording of SLB potentials evoked by laser stimulus. Based on the biopotential records obtained with this method, a scientifically based conclusion can be drawn about the effectiveness of the commercially available devices for low level laser therapy used in Medical Acupuncture. The prototype tests showed that with the biostimulator presented, SLB could be effectively stimulated at low power levels. However more studies are needed to derive a general conclusion about the biostimulation mechanism of lasers in Medical Acupuncture and their most effective power and optical settings.The authors contributed equally to this work