Diseases of the dental pulp and periapical tissues are chiefly caused by microorganisms. Antibiotics are used in some endodontic cases; however, successful cases can predominantly be achieved by mechanical and chemical cleaning of the canal or surgical intervention.The aim of this study was to determine the knowledge of General Dental Practitioners (GDPs) in Shiraz in respect to antibiotic prescriptions during and after endodontic treatment. A one-page questionnaire was sent to 200 active general dentists. Of the 120 surveys returned, 93 were accepted. The data were analyzed using t-test, Chi-square, ANOVA and Fisher’s Exact Test.Only 29% of dentists had full knowledge  of antibiotic prescription protocols in pulpal and periapical disease. Amoxicillin 500 mg capsule was the drug of choice of dentists. Total of 42% of GDPs had full knowledge of antibiotic prescription protocols for persistent or systemic infections cases. GDPs more recently qualified had slightly greater knowledge compared to GDPs with experience; however, this difference was not significant. Also, there was no significant difference between genders.General practitioners’ knowledge about antibiotics seems inadequate and further education is recommended to update the practitioners. Bacterial resistance to antimicrobials has been an ongoing problem for clinicians ever since the discovery of antimicrobial agents; this is due to bacterial species ability to develop resistance to all antibacterial agents shortly after they had been marketed and used .There is sufficient evidence that shows a significant relationship between increase in antimicrobial resistance and antimicrobial utilization, with higher resistance levels in bacteria isolated from areas with high antibiotic utilization compared with those from areas with low antibiotic utilization .Antibiotics can be responsible for various adverse effects, including drug interaction, nausea, gastrointestinal upsets, potentially fatal allergic reactions and antibiotic associated colitis . AntibioPalmer et al. showed that 12.5% of general dental practitioners (GDPs) prescribed antibiotics for teeth with acute pulpitis.Antibiotic prescription has been reported for 30.3% of GDPs in cases of time limitations, and 47.3% when a precise diagnosis was not impossible. The most commonly prescribed antibiotic was amoxicillin . AnotherZadik and Levin evaluated the influence of geographic location of graduation  on decision making regarding management of dental caries, periapical lesions and antibiotic prescribing routines. They found that significantly more Latin American graduates prescribed antibiotics following endodontic treatment, retreatment, and third molar extractions .The analysis of national data available from health solutions in Wales showed that dental prescribing in Wales accounted for 9% of total antibacterial prescribing in primary care in 2008. Penicillin and metronidazole constituted the bulk of antibiotics prescribed by the dentists . A studyMurti and Morse in a study conducted in Fiji found that prescription of antibiotic increased with clinical experience and there was a moderate level of knowledge regarding specific indications for antibiotic prescription both therapeutically and prophylactically. In addition, approximately one third of respondents stated antibiotic resistance as a problem in Fiji and 40% reported experiencing some form of antibiotic resistance in clinical practice .The knowledge and practice of Shiraz general practitioners has not been assessed yet. The aim of this study was to determine GDPs’ knowledge of antibiotic prescribing in Shiraz during and after endodontic treatment.This descriptive research was approved by an ethical committee in Shiraz. A questionnaire  was desiThe study sample was selected from dentists in Shiraz accredited by Islamic Republic of Iran Medical Council. To determine the sample volume a Kokaran formula was used and 10% of GDPs (n=93) were selected through a cluster sampling. Shiraz was divided into five geographic zones. Stratified clustering sampling technique was used which incorporated 5 stratified zones, for each of which a cluster of active general dentists were randomly recruited. Overall, 200 GDPs were recruited to this study and a one page questionnaire was sent to them . A totalThe overall response rate to questionnaires was 46.5 % (93 fulfilled acceptance criteria). The demographics of respondents are described in ANOVA test revealed no significant differences between respondents in relation to sex and number of years in practice. Only 1% of respondents answered all the questions of prophylactic antibiotic coverage for medically compromised patients correctly. The drug of choice for these cases was 2g Amoxicillin (80.4%). Other prophylactic drugs that were prescribed by dentists are summarized in The greatest number of antibiotic prescriptions was written for acute pulpits (80.6%) and acute periapical abscess (74.2). Overall, 11% of respondents always prescribed antibiotics after root canal therapy (11%) . For chrOverall, only 20% of GDPs had full knowledge of antibiotic prescription for pulpal and periapical diseases. Amoxicillin 500mg was the drug of choice (57.6%) in all correct and incorrect cases. Based on the correct answers, 42% of GDPs had full knowledge of antibiotic prescription. In cases of persistent or systemic infection , penicillin IM was the drug of choice (26.6%), followed by amoxicillin 500mg (25.3%).The overall response rate from 200 GDPs in this study was 46.5%. Other similar surveys published have reported response rates of 35% and 31% [20]; theGDPs that had graduated more recently had greater knowledge compared to GDPs with more experience. These results concurred with a similar study performed by Gutierrez et al. . This isAntibiotics are not indicated for acute pulpitis; however, 80.6% of Shiraz GDPs prescribed antibiotics; whereas Palmer et al. showed that 12.5% of British GDPs prescribed antibiotics . AnotherAntibiotics are not indicated and will not assist cases where the pulp is still vital and there are no signs of local or systemic infection/involvement .Even in cases of a necrotic pulp, chronic apical periodontitis with fistula or a chronic periapical lesion in a healthy patient, there is no indication for antibiotic use and treatment should be limited to non-surgical root canal therapy. In this study 58% of GDPs prescribed antibiotics which were very different to percentages quoted in other studies 35.7%  and 18.8GDPs knowledge of dental infections with systemic signs like fever, malaise, difficulty in swallowing was inadequate; only 42% answered all the related questions correctly. Various antibiotics were prescribed in these cases which were similar to reports of other studies 26]27][[27][26][[[27][26]According to the present study, general dentists in Shiraz use antibiotics inappropriately; this could lead to problems such as drug resistance, resistant microorganisms and other side effects.It would appear from this study that GDPs knowledge about the use of antibiotics in general practice is far from ideal. This mirrors general medical practice where studies have shown that decision making in antibiotic therapy requires improvement. Rational prescribing based on a thorough evidence-based knowledge is essential. Effective communication between microbiologists and practitioners and the publication of prescribing guidelines and protocols could help to achieve this . MoreoveThe use of clinical audit and computers along with other tools to increase knowledge of antibiotic prescribing and improve patient care should not be underestimated.This study supports the conclusion that there is a lack of knowledge about the correct indication, type, and dosage of antibiotics in dental practice. There is also a need to improve undergraduate education and to increase the provision of postgraduate courses and other educational initiatives on antibiotic prescribing.


In Ethiopia, more adolescents are in school today than ever before; however, there are no studies that have assessed their comprehensive knowledge of HIV/AIDS. Thus, this study tried to assess the level of this knowledge and the factors associated with it among in-school adolescents in eastern Ethiopia.A cross-sectional school-based study was conducted using a facilitator-guided self-administered questionnaire. The respondents were students attending regular school in 14 high schools located in 14 different districts in eastern Ethiopia. The proportion of in-school adolescents with comprehensive HIV/AIDS knowledge was computed and compared by sex. The factors that were associated with the comprehensive HIV/AIDS knowledge were assessed using bivariate and multivariable logistic regression.Only about one in four, 677 (24.5%), in-school adolescents have comprehensive HIV/AIDS knowledge. The knowledge was better among in-school adolescents from families with a relatively middle or high wealth index , who got HIV/AIDS information mainly from friends or mass media  and who received education on HIV/AIDS and sexual matters at school (adjusted OR [95% CI]=1.59 [1.22–2.08]). The females were less likely to have comprehensive HIV/AIDS knowledge compared to males (adjusted OR and [95% CI]=0.60 [0.49–0.75]).In general, only about a quarter of in-school adolescents had comprehensive HIV/AIDS knowledge. Although the female adolescents are highly vulnerable to HIV infection and its effects, they were by far less likely to have comprehensive HIV/AIDS knowledge. HIV/AIDS information, education and communication activities need to be intensified in high schools. In Ethiopia, a large number of adolescents are enrolled in high schools, and a significant proportion of rural students attend high school away from their home village. The level of comprehensive HIV/AIDS knowledge and access to HIV/AIDS information and services have been matters of great concern . In EthiPrevious studies conducted in Ethiopia revealed that residing in urban areas, higher educational attainment and male gender are positively associated with increased awareness of HIV prevention methods . StudiesIn Ethiopia, there are only a few studies that have assessed the level of the comprehensive HIV/AIDS knowledge of in-school adolescents. The available studies revealed that sexual debut during adolescence is associated with the risk of being HIV positive at later ages and that secondary school adolescents have the highest HIV positive proportion among the youth age groups in Ethiopia ,11. FurtN=2860) was determined by OpenEpi web-based epidemiological calculator based on the assumptions of 95% significance level; 25% males and 19% females had the outcome =0.60 [0.49–0.75]). Family wealth index was associated with comprehensive HIV/AIDS knowledge, in that adolescents from a middle or high family wealth index were more likely to have comprehensive HIV/AIDS knowledge compared to those from a low family wealth index . The family wealth index effect was stronger and significant for adolescents from families in rural areas compared to those from families in urban areas . The major sources of information on HIV/AIDS were associated with comprehensive HIV/AIDS knowledge. Adolescents who reported friends or mass media as their major sources were more likely to have comprehensive HIV/AIDS knowledge compared to those who cited family members as their major source . Adolescents who reported that they had been taught about HIV/AIDS and the related topics at school were 1.59 times more likely to have comprehensive HIV/AIDS knowledge compared to those who did not report being taught on such topics (adjusted OR [95% CI]=1.59 [1.22–2.08]). Discussion on sexual matters with parents or other family members was not associated with comprehensive HIV/AIDS knowledge (adjusted OR [95% CI]=1.01 [0.81–1.25]) .Only about a quarter of the in-school adolescents had comprehensive HIV/AIDS knowledge. The knowledge was more common among in-school adolescents from families with a middle or higher wealth index, who got HIV/AIDS information mainly from friends or mass media and who received HIV/AIDS and sexual matters education at school. Although the females are highly vulnerable to HIV infection and its effects, they were less likely to have comprehensive HIV/AIDS knowledge compared to males. They were also less likely to have comprehensive pregnancy knowledge, even though they had more knowledge on pregnancy occurrence dates related to the menstrual cycle.The major source of bias in this study might emerge from the self-administered data collection technique in which respondents might have failed to understand the questions correctly. To overcome this bias, data were collected by a facilitator-guided self-administered method . The respondents were also provided with questionnaires prepared in all possible languages respondents might understand well. Even though it may be difficult to totally overcome the bias which arises from such methods of data collection, its effect on the findings of this study is negligible.The level of comprehensive HIV/AIDS knowledge in this study was lower than the previous AIDS awareness and prevention strategy knowledge estimates . The reaComprehensive HIV/AIDS knowledge was associated with the sex of the respondents. The females were less likely to have comprehensive HIV/AIDS knowledge compared to the males. This finding is consistent with the Ethiopian DHS report on HIV prevention strategy knowledge and previous in-school adolescents study which reported low HIV transmission modes knowledge among females ,4,10. ThFamily wealth index was associated with comprehensive HIV/AIDS knowledge. The adolescents from middle or high family wealth index were more likely to have comprehensive HIV/AIDS knowledge compared to those from a low family wealth index. This is consistent with a finding from another study which reported an increase in mean-knowledge score by increasing socio-economic class . This maThose who cited friends and mass media as their major sources of HIV/AIDS information were more likely to have comprehensive HIV/AIDS knowledge compared to those who reported their parents or other family members as their major sources. This was not consistent with other study findings . This isAttending classes on HIV/AIDS and sexual matters at school was significantly associated with comprehensive HIV/AIDS knowledge, in that the respondents who reported that they had attended such classes were more likely to have comprehensive HIV/AIDS knowledge compared to those who did not attend such classes. This may be because, even though such topics were integrated into some subjects in schools, some schools and/or teachers may not teach these topics as they might feel they are not well trained on the topic, while some other schools and teachers may teach such topics by making extra effort themselves or by inviting other relevant professionals.Discussing sexual matters with parents or other family members was not associated with comprehensive HIV/AIDS knowledge. This finding is not consistent with the report of another study . This coIn conclusion, only about one in four of the in-school adolescents had comprehensive HIV/AIDS knowledge. The factors associated with comprehensive HIV/AIDS knowledge of in-school adolescents were both individual factors (sex) and contextual factors . Although the female adolescents are highly vulnerable to HIV infection and its effects, they were by far the less likely to have comprehensive HIV/AIDS knowledge. Thus, HIV/AIDS information, education and communication activities need to be intensified in high schools, including further attention being put on gender, the family wealth disparity, the positive influences of peers, mass media and teaching methods of HIV/AIDS and related issues at schools.


However, patients with hyper-interleukin (IL-) 6 syndromes such as multicentric Castleman's disease, rheumatoid arthritis, and other immune-mediated conditions frequently show lymph node involvement and often fulfill the diagnostic criteria for IgG4-RD. Owing to these factors, IgG4-RD cannot be differentiated from hyper-IL-6 syndromes on the basis of histological findings alone. Laboratory analyses are crucial to differentiate between the 2 diseases. Hyper-IL-6 syndromes are characterized by elevated serum levels of IgG, IgA, IgM, and C-reactive protein (CRP); thrombocytosis; anemia; hypoalbuminemia; hypocholesterolemia. In contrast, IgG4-RD does not share any of these characteristics. Therefore, the diagnosis of IgG4-RD requires not only pathological findings but also clinical and laboratory analyses.Lymphadenopathy is frequently observed in patients with immunoglobulin G4-related disease (IgG4-RD) and sometimes appears as the first manifestation of the disease. The diagnosis of IgG4-related lymphadenopathy is complicated owing to a great histological diversity, with at least 5 histological subtypes. Indeed, lymph node biopsy may be performed under the suspicion that the lymphadenopathy is a malignant lymphoma or other lymphoproliferative disorder. The diagnosis of IgG4-RD is characterized by both elevated serum IgG4 (>135 mg/dL) and histopathological features, including a dense lymphoplasmacytic infiltrate rich in IgG4 Immunoglobulin G4-related disease (IgG4-RD) frequently involves lymph nodes in a localized or systemic fashion –3. Indee+ plasma cells; an IgG4+/IgG+ plasma cell ratio of >40% is an important diagnostic criterion for the disease [IgG4-RD is an inflammatory condition characterized by a dense lymphoplasmacytic infiltrate rich in IgG4 disease , 5. Pati disease –3, 5, 6. disease –3, 7, 8. disease , 3.Recently, several studies dealing with the morphological and immunohistological findings of IgG4-related lymphadenopathy have been performed –3. Furth+ plasma cell infiltration  and elevated serum IgG4 levels [This type is frequently characterized by systemic lymphadenopathy –3. Histo4 levels . TherefoThe lymph nodes usually exhibit reactive follicular hyperplasia, and sinuses are intact. The reactive follicles comprise a germinal center surrounded by a discrete mantle zone. The interfollicular zone contains a small to moderate number of mature plasma cells, with small lymphocytes and eosinophils . This tyThis type is also frequently characterized by systemic lymphadenopathy –3. Histo+ follicular dendritic cell proliferation, the presence of CD10+ T-cells, and T-cell receptor gene rearrangement are not observed [This type is somewhat similar to angioimmunoblastic T-cell lymphoma. However, it is noteworthy that these lesions lack clusters of clear cells and definite cytologic atypia typical of the lymphoma. Moreover, CD21observed .+ plasma cells in the germinal centers, with only a small number present in the interfollicular zone [+ plasma cells are detected in both the germinal centers and interfollicular zone [PTGC is a benign condition of unknown origin characterized by reactive follicular hyperplasia in the lymph nodes , 11. Reclar zone . Howeverlar zone . Patients with this type have a uniform clinicopathology. The patients initially present with asymptomatic localized submandibular lymphadenopathy, with half of them showing progression to extranodal IgG4-RD, systemic disease, or both during the follow-up period .In this type, the lymph nodes show asymptomatic localized lymphadenopathy . Histolo These histological findings are somewhat similar to those characteristic of nodal IPT. Nodal IPT has been histologically classified into 3 stages  , 15. IPT Hyper-IL-6 syndromes such as MCD, RA, and other immune-mediated conditions are characterized by elevated serum IL-6 levels , 17. MorUnlike IgG4-RD that involves other organs, IgG4-related lymphadenopathy shows histological diversity, with 5 distinct subtypes. Moreover, recently, Takahashi et al. reported a unique case of IgG4-related lymphadenopathy with epithelioid granuloma . This hiIndeed, hyper-IL-6 syndromes can often fulfill the diagnostic criteria for IgG4-RD. Therefore, IgG4-RD, and especially IgG4-related lymphadenopathy, cannot be differentiated on the basis of histological findings alone. The diagnosis of IgG4-RD needs to be based not only on pathological findings but also on clinical and laboratory findings.


Because economic data on the prophylactic usage of antibiotic in Iran are scant, we have conducted a cross-sectional study with provider perspective to measure costs and appropriate use of antibiotics in surgical wards of 6 training hospitals affiliated to Shiraz University of Medical Sciences (SUMS), Iran.Over a six-month period 1,000 consecutive patients undergoing surgical operation were enrolled and information on prophylactic antibiotic administration was collected. The information included basic patient's demographic data, types of surgery, category of antibiotic, dosage, dosage intervals, route of administration, number of doses, initiation times and duration of administration. In order to determine the agreement between prescribed antibiotics and medical indication, the American Society of Health-System Pharmacists (ASHP) guidelines were applied.Nine hundred and ninety three out of 1,000 patients (99.3%) had received at least one antibiotic and 908 patients (91.4 %) received antibiotics because of a medical indication. Five out of 913 patients who had indications for antibiotic prophylaxis did not receive any antibiotic. Antibiotics were prescribed for 85 out of 87 (98%) procedures in which an antibiotic was not indicated. The average cost of antibiotic prescription per surgical procedure was 786,936 Iranian Rials (corresponding to 99.60 USD or €82.90). The most frequent prescribed antibiotic was cefazoline adding 53.3% of the total cost of antibiotics. In total, 36,516,190 Iranian Rials  were spent for cefazoline alone.The results of this study showed that all surgical patients received at least one antibiotic as prophylaxis for any infection in the surgical site. Our results indicate over- and misuse of antibiotics in Iran leading to a great amount of economic burden, since in 98% of all procedures, antibiotics were used inappropriately. Without any doubt in course of infectious diseases, antibiotics can have an important role in global reduction of morbidity and mortality. However, if mis- or overused, they would result into an emergence for resistance to bacteria and lead to unnecessary costs. BacteriaIn several hospital settings in the US and Europe, antibiotics were shown as the second prescribed therapeutic drugs. 60-90% of patients receive antibiotics, and in 40% of the cases, prescriptions are often without any laboratory confirmation or have any indication for antibiotic use. It was shown that the rate of antibiotic misuse are higher in surgical departments than in internal medicine ones, and were mostly for prophylaxis than treatment.In developing countries, antibiotics are the largest single group of drugs purchased by patients. At the same time, in many developing countries, the availability and use of antibiotics are poorly controlled and of course, it has been demonstrated that a restriction policy can be effective in decreasing the antibiotic use and increasing the rational prescription. Misuse oThere may be many explanations for this trend, including the patients’ wrong attitude in using excessive drugs and doctors’ tendency towards satisfaction of their patients though prescribing the requested medications. On the oBecause of mis- and overuse of antibiotics to prevent any infection in the surgical site in Shiraz, southern Iran8]9] an[9] an8][ an[9] anFrom February to July 2004 in surgical wards of 6 teaching hospitals affiliated to Shiraz University of Medical Sciences, Iran, similar to previous studies,8]9] 1,[9] 1,8] 1,In order to determine the agreement between prescribed antibiotics and surgical treatment, the American Society of Health-System Pharmacists (ASHP) guidelines were applied. The collAmong 1,000 consecutive surgical patients, 120 (12.0%) were in departments of Cardiosurgery, 112 (11.2%) in General/Gastrointestinal, 110 (11.0%) in Neurosurgery, 111 (11.1%) in Gynecology, 110 (11.0%) in Ophthalmology, 115 (11.5%) in Orthopedy, 111 (11.1%) in ENT , and 110 (11.0%) in Urology Department. One-hundred and one patients (10.1%) were transplanted patients, among them 23  were kidney, and 78  liver transplanted ones.The patient’s age range was 86 years (1 to 87) (mean: 40±22). More than half of the patients were male (56%), and most of the procedures (91.4%) were elective. Almost all patients  had at least one prescribed antibiotic, and 908 patients (91.4%) received antibiotics because of a medical indication. Five of 913 patients who had indications for antibiotic prophylaxis did not receive any antibiotic. However, an antibiotic was prescribed for 85 out of 87 (98%) procedures in which an antibiotic was not indicated .The costs of antibiotics used for various types of surgeries are shown in Diagram 1. The average cost of antibiotic prescription per surgical procedure was 786,936 Iranian Rials (corresponding to 99.60 USD or €82.90). The most frequent prescribed antibiotic was cefazoline  which waThe quantity/ (DDD/strength)/ 1000 patients= number of DDDs per user per year which was as follows: Cefazolin=7.8175 g, Gentamicin=0.11 g, Amikacin=0.3477 g, Cephalexin=1.181 g, Ampicillin=0.06 g, Ceftriaxone=0.378 g, Cefixime=0.031 g, Chloramphenicol=0.15 g, Ciprofloxacin=0.311 g, Metronidazole=0.23 g, Amoxicillin=0.06 g, Cloxacillin=0.01 g, Clindamycin=0.02 g, Erythromycin=0.004 g, Vancomycin=0.017 g and Penicillin G=97500 units.The results of this study showed that almost all surgical patients received at least one antibiotic as prophylaxis for any infection in the surgical sites. Although this reflects the surgeons’ awareness of the value of antibiotics in prevention of surgical site infections, our results also showed the overuse of antibiotics in Iran, since in 98% of all procedures antibiotics were used inappropriately. This finding is consistent with other studies conducted in average income countries.12]12]Bugnon-Reber and colleagues pointed out that the lack of indication is a popular reason for inappropriate use of antibiotic. It was aOveruse of antibiotics together with improper management may cause an increase in direct cost of treatment. Furthermore, overuse and inappropriate use of antibiotics lead to an increase in both bacterial resistance and cost of treatment. Our findings showed that the rate of consuming an antibiotic costs 4.75 times as much as that recommended by ASHP guideline. Because of inappropriate usage, the six hospitals under study paid an extra expense of 54,054,210 Iranian Rials  for surgical procedures among 1,000 patients.A number of studies have focused on the issue of increasing cost of antibiotics based on inappropriate usage. Parret et al. showed tFraser et al. showed that application of specific guidelines for prescribing antibiotics can lead to an saving up to 400 USD per antibiotic prescription. PestotniOur study has several limitations. As it is important to mention that there are different factors that affect on an appropriate prescription, including cultural and educational factors, nurses and pharmacists influences, distribution and availability of medical compounds, and logical calculations. The influence of these factors was not examined in our study. In order to ensure cost-effective application of prophylactic antibiotics, Ristic´ et al. showed a significant change in prescribing patterns of antibiotic prophylaxis in caesarean sections following introduction of local guidelines. There was a significant decrease in use of ‘third’ generation of cephalosporin’s whereas the use of ‘‘older’’ antimicrobial drugs with better safety and efficacy increased.Therefore, we think that it would be necessary to design an evidence based guideline according to local conditions and cultural background in order to improve patient safety and decrease direct costs in the administration of antibiotic prophylaxis to prevent surgical site infection in Shiraz, Iran.


The proposed MZI sensors are capable of in situ detection in many areas of interest such as environmental management, industrial process control, and public health.A fiber inline Mach-Zehnder interferometer (MZI) consisting of ultra-abrupt fiber tapers was fabricated through a new fusion-splicing method. By fusion-splicing, the taper diameter-length ratio is around 1:1, which is much greater than those (1:10) made by stretching. The proposed fabrication method is very low cost, 1/20–1/50 of those of LPFG pair MZI sensors. The fabricated MZIs are applied to measure refractive index, temperature and rotation angle changes. The temperature sensitivity of the MZI at a length of 30 mm is 0.061 nm/°C from 30–350 °C. The proposed MZI is also used to measure rotation angles ranging from 0° to 0.55°; the sensitivity is 54.98 nm/°. The refractive index sensitivity is improved by 3–5 fold by fabricating an inline micro–trench on the fiber cladding using a femtosecond laser. Acetone vapor of 50 ppm in N AmongHigh-order cladding modes are excited in fiber MZIs, which are guided by the cladding-ambient interface and directly exposed to the environment. The refractive index change of the environment can significantly change the effective propagation constant of the cladding modes. Thus, fiber MZIs can be used as a refractive index sensor by testing the phase shift of interference fringe. Fiber MZIs are also used to detect temperature, curvature, strain and stress ,7,12. ItRecent developments in femtosecond laser technology have opened new possibilities for one-step fabrication  of micro/nano-scale three-dimensional (3D) structures in various solid materials with greatly reduced collateral damage. Especially, new opportunities for laser micromachining of fiber optics devices are possible using ultrashort laser pulses. By using femtosecond lasers, various structures have been fabricated in fibers and used in the sensing field –18.2 after coated with zeolite thin film. In addition, temperature and rotation angle tests are also conducted.Among the various types of MZIs, fiber taper-based structures have the advantages of simplicity and high repeatability ,6. To ob2.in) is split into two portions in the first ultra-abrupt taper: one (I1) through the core and the other (I2) through the cladding. Part of the light (I1) in cladding modes is coupled back into the core at the second ultra-abrupt taper. Interference between the light traveling in the cladding and that traveling in the core is detected by measuring Iout.L is the interferometer length; k is an integer; vλ is the maximum attenuation wavelength of the kth order; The interference signal reaches minimum when the phase difference between cladding and core modes satisfies the following condition :(1)2π : (a) the two normal cleaved ends are made ellipsoidal by a one time discharge  and an optical power meter (Agilent 8163B) is employed to measure the transmission spectra by wavelength sweeping. The tunable laser scans through its wavelength range  at the rate of 5 pm per step. The transmission spectra of fiber in-line MZI with different lengths are shown in 4.4.1.extn is the external refractive index; L is the interferometer length; k is an integer; vλ is the maximum attenuation wavelength of the kth order; The effective refractive index of the cladding mode changes with the variations of external refractive index, and it results in a shift of the maximum attenuation wavelength. The sensitivity can be expressed as :(2)S = a, b, c and d in et al. [The MZI with a length of 40 mm is tested as a refractive index sensor. Salt solutions with various concentrations  are used in the experiments. The corresponding RIs are 1.3330, 1.3366, 1.3400, 1.3435, 1.3470, 1.3505, 1.3541, 1.3576, and 1.3612, respectively . Four atn et al. , higher n et al. .4.2.Due to the difference between the effective refractive index changes of cores and those of claddings in response to the temperature variations, the MZI can be used as a temperature sensor. In a muffle furnace, a 30 mm long MZI is used to test temperature sensitivity. The temperature changes every 5 min from 30 °C to 350 °C with a step of 20 °C. 4.3.In this paper, the proposed MZI is used to measure angular displacement by fixing it on a micro-rotation stage. The corresponding structural diagram is shown in θ. By recording the wavelength shift in the transmission spectrum, we can test the angular displacement. An MZI with length of 25 mm is chosen to test the angular-displacement sensitivity in the angle range from 0° to 0.55° with an increment of 0.05° for a total of 11 steps. θ. The sensitivity is 54.98 nm/° with good linearity. At a resolution of 1pm of the detecting system, the detection limit is 3.17 × 10−7 rad. Compared to other methods [Because of the macro-bending, the coupling of the core mode to the cladding modes is changed by the variation of angle  methods , the pro4.4.In order to improve the RI sensitivity of the proposed MZI sensor, an inline side-ablated micro-trench was fabricated to remove part of the fiber cladding using a femtosecond laser. The micro-trench lies in the center of the MZI, as shown in L = 40 mm) to improve the RI sensitivity. h) of 18 μm and a length (0L) of 200 μm, respectively. The width of 88 μm is obtained accordingly for the fiber is cylinder. The measured salt solutions are the same as in session 4.1. Salt solutions with various concentrations  are used in the experiments. The corresponding RIs are 1.3330, 1.3348, 1.3383, 1.3418, 1.3453, 1.3488, 1.3523 and 1.3558, respectively [a, b, c and d in A side-ablated micro-trench is fabricated in the aforementioned MZI  molecules from the ambient for highly sensitive detection. In this paper, MFI-type zeolite thin film is synthesized on the MZI for detection of acetone vapors with high sensitivity. The zeolite film is coated on the MZI cylindrical surface by given in .The sensor measure gas concentrations by monitoring the molecular adsorption-induced changes of the interference pattern position. The sensitivity is affected by many parameters such as the type of the zeolite, the film thickness, the measurands and the characteristics of the MZI. The length of the MZI is 36 mm, while the transmission spectrum is changed completely after being coated with zeolite film  achieved by stretching. The fabricated MZIs are applied to sense refractive index, temperature and rotation angle changes. The temperature sensitivity is 0.061 nm/°C in 30–350 °C. At a resolution of 1 pm of the detecting system, a detection limit is 3.17 × 10


Saccharina latissima (SL), Fucus vesiculosus (FV), and Cladosiphon okamuranus (CO), two chemically modified fucoidans with a higher degree of sulfation , and a synthetic totally sulfated octasaccharide (OS), related to fucoidans, were assessed on anticoagulant and antithrombotic activities in different in vitro experiments. The effects were shown to depend on the structural features of the compounds tested. Native fucoidan SL with a degree of sulfation (DS) of 1.3 was found to be the most active sample, fucoidan FV (DS 0.9) demonstrated moderate activity, while the polysaccharide CO (DS 0.4) was inactive in all performed experiments, even at high concentrations. Additional introduction of sulfate groups into fucoidan SL slightly decreased the anticoagulant effect of SL-S, while sulfation of CO, giving rise to the preparation CO-S, increased the activity dramatically. The high level of anticoagulant activity of polysaccharides SL, SL-S, and CO-S was explained by their ability to form ternary complexes with ATIII-Xa and ATIII-IIa, as well as to bind directly to thrombin. Synthetic per-O-sulfated octasaccharide OS showed moderate anticoagulant effect, determined mainly by the interaction of OS with the factor Xa in the presence of ATIII. Comparable tendencies were observed in the antithrombotic properties of the compounds tested.Three structurally different fucoidans from the brown seaweeds  Thromboembolic disorders, such as venous thromboembolism and arterial thrombosis, are considered to be among the main reasons for cardiovascular diseases. The situation leads to increased utilization of anticoagulant and antithrombotic agents in medical practice. Heparin and its derivatives are usually recommended as support therapy ,2, but sNatural sulfated polysaccharides, such as galactans ,4,5,6,7,A number of studies revealed certain structure-activity relationships for these macromolecules ,14,15,16O-sulfation together with the structure of the backbone and branches, but not only total negative charge of sulfates, has a substantial impact on the activity of the biopolymers under discussion (see for example [O-sulfated (1→3)-linked α-l-galactan, but not an α-l-fucan, with a similar sulfation pattern and molecular size, is a potent thrombin inhibitor mediated by ATIII or HCII [O-sulfated fucose units [l-fucans, enriched in 2,4-di-O-sulfated units, were shown to have an amplifying effect on the ATIII-mediated anticoagulant activity [The pattern of  example ). Thus,  or HCII . In the se units . In addiactivity .Fucus vesiculosus and Laminaria brasiliensis, which are built up of alternating (1→3)- and (1→4)-linked α-l-fucose residues sulfated at C-2 and/or C-4, and bearing fucose branches [Saccharina latissima (previous name Laminaria saccharina) and Fucus distichus, but polysaccharides from Cladosiphon okamuranus and Analipus japonicus were inactive [l-arabinose residues sulfated at C-2 and/or C-4 was also found to be the potent direct thrombin inhibitor [Fucoidans from brown algae possess a significantly more complicated structure than fucans from invertebrates due to the presence of numerous branches, non-fucose monosaccharide constituents, and acetates . It was branches . The siminactive . Notablynhibitor .O-sulfated fucoidan derivatives and structurally related synthetic octasaccharide were studied as well.In this paper we report the results of the study on anticoagulant and antithrombotic activities of three fucoidans from brown seaweeds differed in the monosaccharide composition, types of glycosidic bonds, sulfate content, and sulfation pattern. Chemically S. latissima -α-l-fucopyranosyl backbone, sulfated at C-4 and branched at C-2. Its degree of branching is comparable with that of SL, but this fucoidan contains single α-d-glucuronic acid residues as branches instead of fucose residues [Three samples of native fucoidans were used in this study . Their monosaccharide composition and sulfate content are summarized in 1.25” in ), which 1.25” in , containa branch  -α-l-fucoside OS  was chosen as a reference, as this polysaccharide is intensively used in medical practice as a heparinoid anticoagulant with low risk of side effects [® were compared.General clotting assays were performed with the use of normal plasma, which was incubated with the samples. Commercially available, low-molecular-weight heparin Clexane effects ,25,26. TTo evaluate the influence of the fucoidans and their derivatives on the intrinsic pathway of coagulation, the activated partial thromboplastin time (APTT) assays have been performed. The dose-depended changes in the APTT value are shown on ®. Double increasing of control value of APTT was achieved at a concentration of ~1.1 μg/mL for SL and ~3.3 μg/mL for Clexane®. Fucoidan FV showed moderate activity (2APTT ~6.8 μg/mL), while polysaccharide CO was inactive even at a concentration of 100 μg/mL.The effect on blood coagulation was shown to depend on the structural features of the tested sample. Among parent fucoidans, the polysaccharide SL demonstrated the highest level of activity, even exceeding that of ClexaneO-sulfation of inactive fucoidan CO gave product CO-S , indicating that its molecular size is not sufficient for achieving a high effect in this test.Interestingly, an opposite situation was observed in the case of the samples SL and SL-S. Additional introduction of sulfates into the structure of SL led to a slight decrease in the anticoagulant effect; however, the sample SL-S (2APTT~2.0 μg/mL) was slightly more active than CO-S. It should be noted that synthetic per-To assess the influence of the studied compounds on the extrinsic pathway of coagulation, the value of Prothrombin time (PT) was measured. All samples, except CO, slightly increased PT in a dose-dependent manner  within the concentration range used in the APTT test. According to literature data , signifi® (~2.2 μg/mL), SL (~2.4 μg/mL), and SL-S (~2.2 μg/mL) suggested that these preparations possess comparable effects. Increasing polysaccharide concentration gave quite different responses. Thus, at a concentration of ~3.8 μg/mL Clexane® prolonged blood coagulation by eight times, while SL and SL-S gave prolongation only by four and three times, respectively. It is noticeable that, similarly to the results of the APTT test, the chemically sulfated polysaccharide SL-S exhibited slightly lower activity than parent fucoidan SL.The influence of the samples on thrombin-induced clot formation was also investigated. The value of Thrombin time (TT) was measured at several concentrations see  to againThe fucoidan CO was inactive in the TT assay even at a concentration of 100 μg/mL, while its sulfated derivative CO-S at a concentration of ~7.5 μg/mL demonstrated a comparable effect with preparation SL-S at the same concentration. Significant activity of the samples SL, SL-S, and CO-S in this test could be explained by their ability to interact with thrombin. Similarly to the results of the APTT test, fucoidan FV showed moderate inhibitory effect in TT assay . This can be due to its slightly lower degree of sulfation 0.9,  in compa®, another heparin-related drug, Arixtra® , was used as a reference. The results are shown in To investigate further the mechanism of anticoagulant action of fucoidans and their derivatives, the experiments with purified proteins were performed. These studies were based on the assay of amidolytic activity of thrombin (IIa) or factor Xa using chromogenic substrates, as described previously ,14,15. T® was active only in the presence of ATIII. Effectiveness of binding to thrombin in the presence of ATIII was high for the samples SL, SL-S, CO-S, and Clexane® (IC50 < 1.0 μg/mL), but it was slightly lower for the fucoidan FV (IC50 ~2.1 μg/mL). The fucoidan CO was inactive in all tested concentrations.The preparations SL, SL-S, CO-S, and FV were shown to interact with thrombin both in the presence and in the absence of ATIII, while heparinoid Clexane® did not inhibit thrombin activity in the presence of ATIII even at a concentration of 100 μg/mL, while the polysaccharide Clexane® demonstrated significant inhibitory effect in this experiment (IC50 < 1.0 μg/mL). It was established earlier [The synthetic octasaccharide OS demonstrated more than 100 times lower anti-IIa activity in the presence of ATIII than the polysaccharides SL, SL-S, and CO-S, with the same backbone. This result could be compared with known data for heparinoids. Thus, synthetic pentasaccharide Arixtra earlier ,28,29 thFor direct thrombin inhibition, higher concentrations of polysaccharides were required. Thus, the samples SL, SL-S, and CO-S showed 50% inhibition at concentrations of ~45.9, ~55.9, and ~58.9 μg/mL, respectively, while the fucoidan FV demonstrated only 25% inhibition at a concentration of 59.0 μg/mL.l-fucoside OS, were found to be unable to interact directly with thrombin. It is interesting to mention that, according to the theoretical prediction, the high activity of linear sulfated (1→3)-β-l-arabinan was explained by the specific structure of its octasaccharide fragment [The fucoidan CO, as well as the synthetic octa-(1→3)-α-fragment . The dif50 were ~1.1, ~1.9, and ~2.0 μg/mL, respectively), but their activity was significantly lower than that for heparinoids Clexane® and Arixtra® . The fucoidan FV showed 50% inhibition only at a concentration of ~28.0 μg/mL. Surprisingly, the synthetic per-O-sulfated octasaccharide OS demonstrated moderate anti-Xa activity in the presence of ATIII (IC50 were ~13.0 μg/mL), even exceeding the effect of polysaccharide FV. Neither fucoidans, nor heparinoids, bind to factor Xa in the absence of ATIII (data not shown).The polysaccharides SL, SL-S, and CO-S efficiently bind to factor Xa only in the presence of ATIII -linked α-l-fucose units demonstrated high effects in clotting assays and also possessed significant anti-IIa and anti-Xa activities. These results are in good agreement with the data obtained previously for linear highly sulfated fucans from invertebrates [The tendencies found in amidolytic experiments . Two types of inducers of platelets aggregation, namely thrombin and adenosine diphosphate (ADP), were used. All samples were tested at a concentration of 100 μg/mL, while the fucoidan SL was also studied at a concentration of 10 μg/mL .®, efficiently inhibited thrombin-induced platelets aggregation. The 51% inhibition was observed for SL, 48% for SL-S, 42% for CO-S, and 88% for Clexan®, while the fucoidans FV and CO, the synthetic octasaccharide OS, as well as SL (at a concentration of 10 μg/mL), were inactive in this test. Thrombin is considered as the main protein target in this assay and, thus, the results obtained demonstrate comparable tendencies described above for the amidolytic assay.The polysaccharides SL, CO-S, and SL-S, as well as heparinoid Clexane®. Contrary to the mechanism of thrombin-induced platelets aggregation, the polysaccharide inhibitors interact directly with special receptors on the surface of platelets but not with the inducer of aggregation. That is why the observed values of inhibitory effects of the tested samples differed from those measured in the previous experiment. Inhibition of aggregate formation in PRP was 50% for SL-S, 32% for CO-S, and 22% for SL. The fucoidans FV and CO, the synthetic octasaccharide OS, as well as SL (at a concentration of 10 μg/mL) were inactive again.The preparations SL, SL-S, and CO-S demonstrated a statistically significant effect on ADP-induced platelets aggregation exceeding that for ClexaneS. latissima was described earlier [F. vesiculosus was purified by decoloration with NaClO2 in dilute HCl [Preparation of the fucoidan SL (“fraction F-1.25” in ) from th earlier . A crudelute HCl  followedlute HCl , giving O-sulfation under acid-promoted conditions [l-fucose [1H and 13C NMR spectroscopy.The fucoidans SL and CO were subjected to nditions , transfol-fucose  and charThe monosaccharide composition and sulfate content of the samples were determined as described earlier  and summ® hemostasis analyzer  according to the established procedure and using standard reagents (Diagnostica Stago). A solution (15 μL) containing 15 μg, 7.5 μg, or 3.75 μg of a sulfated carbohydrate sample in saline solution (0.9% NaCl) was added to the normal platelet-depleted citrated plasma (3 mL). Low-molecular-weight heparin Clexane® (manufactured by Sanofi) was used for the comparison of anticoagulant activity of the samples. The saline solution was used as a control. The samples were incubated at 37 °C for 1 min.APTT and PT were determined with an SRA-R EvolutionA kit, “Thrombin test” , was used for determination of thrombin time. A saline solution with a sulfated carbohydrate sample (20 μL) was added to the normal plasma (80 μL). The mixture was incubated at 37 °C for 1 min, and a saline solution (100 μL) of stabilized thrombin (10 U/mL) was added. The saline solution was used as a control. The time of clot formation was then determined.para-nitroaniline was measured at 405 nm on an Ultospec II spectrophotometer .Determination of amidolytic activity of thrombin was performed using “ReaChrom ATIII test” kit  as described previously . A solutDetermination of amidolytic activity of factor Xa was performed using “ReaChrom Heparin”  kit. A solution of ATIII  or the buffer (50 μL) was added to a solution of a sulfated carbohydrate sample (0.0001–10 μg) in the buffer . Then an aqueous solution of the factor Xa  was added. The mixture was worked-up, treated with chromogenic substrate, and analyzed as described above.Blood with citrate buffer (9:1) was centrifuged at 1000 rpm for 5 min, and platelets rich plasma was collected from different tubes and combined. A saline solution (10 μL) containing 45 μg of a sulfated carbohydrate sample was added to 450 μL of this plasma. The saline solution was used as a control. The samples were incubated at 37 °C for 2 min, then 10 μm of ADP or 0.5 U of thrombin was added, and light transmittance was measured on a Chrono-Log aggregometer. The data were transformed to percent of platelets aggregation using the established software. The control was considered as 100% of platelets aggregation.n = 4). The results are presented as mean ± S.D. Statistical significance was determined with Student’s t test. The P values less than 0.05 were considered as significant.All experiments were performed in quadruplicate (O-sulfated (1→3)-linked α-l-fucose units demonstrated high effect in clotting assays, while the fucoidan FV built up of alternating (1→3)- and (1→4)-linked α-l-fucose residues with a degree of sulfation of 0.9 was remarkably less active. The polysaccharide CO with the (1→3)-linked α-l-fucose backbone containing α-d-glucuronic acid branches at C-2 with the degree of sulfation 0.4 did not influence on blood coagulation, and this feature could be explained by the absence of a sufficient amount of sulfate groups in its structure, or by the presence of glucuronic acid side residues. The tendencies found in clotting assays correlated with the results obtained in experiments with individual proteins. High anti-IIa and anti-Xa activities were shown for the samples SL, SL-S, and CO-S in the presence of ATIII. The fucoidan FV demonstrated moderate effects under these conditions, while the fucoidan CO was inactive. It is noticeable that the branched polysaccharides with the degree of sulfation ≥0.9  were also shown to be direct thrombin inhibitors and, hence, they differ from heparinoids and linear fucans from invertebrates. The synthetic per-O-sulfated octasaccharide OS, which was structurally related to the polysaccharides SL, SL-S, and CO-S, possessed a moderate effect on clot formation connected mainly with its moderate anti-Xa activity. This result indicated that longer, and probably branched, fucoidan fragments are required for the efficient inhibition of blood coagulation. The most active samples SL, SL-S, and CO-S also exhibited a statistically significant inhibitory effect on platelets aggregation mediated by ADP and thrombin.The effect of the fucoidans on blood coagulation and platelets aggregation was shown to depend on their structural features. Thus, the branched polysaccharides SL, SL-S, and CO-S, enriched in 2,4-di-


Background: Fab arm exchange requires weak interactions between CH3 domains, such as in human IgG4.Results: CH3-CH3 interactions differ >1,000,000-fold between human subclasses and allotypes due to variations Lys/Asn-392, Val/Met-397, and Lys/Arg-409.Conclusion: For IgG2 and IgG3, but not IgG1, hinge disulfide bonds are essential to prevent half-molecule dissociation.Significance: Subclass/allotype variation in the CH3 domain can alter antibody stability and functionality. in vivo for IgG4.Interdomain interactions between the CH3 domains of antibody heavy chains are the first step in antibody assembly and are of prime importance for maintaining the native structure of IgG. For human IgG4 it was shown that CH3-CH3 interactions are weak, resulting in the potential for half-molecule exchange (“Fab arm exchange”). Here we systematically investigated non-covalent interchain interactions for CH3 domains in the other human subclasses, including polymorphisms , using real-time monitoring of Fab arm exchange with a FRET-based kinetic assay. We identified structural variation between human IgG subclasses and allotypes at three amino acid positions  to alter the strength of inter-domain interactions by >6 orders of magnitude. Each substitution affected the interactions independent from the other substitutions in terms of affinity, but the enthalpic and entropic contributions were non-additive, suggesting a complex interplay. Allotypic variation in IgG3 resulted in widely different CH3 interaction strengths that were even weaker for IgG3 than for IgG4 in the case of allotype G3m, whereas G3m(s*/15*) was equally stable to IgG1. These interactions are sufficiently strong to maintain the structural integrity of IgG1 during its normal life span; for IgG2 and IgG3 the inter-heavy chain disulfide bonds are essential to prevent half-molecule dissociation, whereas the labile hinge disulfide bonds favor half-molecule exchange  A). A molecule of IgG contains two Fab arms, which include the first two domains of a heavy chain and an entire light chain. These parts of the molecule contain the complementarity determining regions that are involved in antigen binding. The remaining parts of the two heavy chains form the Fc part, responsible for antibody effector functions, by interacting both covalently in the “hinge region” and non-covalently in their CH3 domains, linking both half-molecules into one antibody molecule. These interactions are therefore of prime importance for the folding, structural integrity, and functionality of the molecule. In addition, the CH2 domains contain N-linked glycans that are important for the correct folding of the Fc part and influence Fcγ receptor binding and complement activation  antibodies, the most abundant class of immunoglobulins, are composed of two heavy and two light chains, each comprising two or four immunoglobulin domains, respectively A. A moletivation , 42.A). Consequently, these antibodies are unable to cross-link antigen to form immune complexes. This process has been observed only for IgG4 and not for IgG1; IgG2 and IgG3 have not been examined thus far. A single amino acid difference between the CH3 domains of IgG1 (Lys-409) and IgG4 (Arg-409) was found to be responsible for facile dissociation of the latter compared with no observable dissociation of the former .Human IgG4 is a striking example of altered functionality as a consequence of altered inter-heavy chain interactions. Due to relatively labile disulfide bonds in the hinge as well as weak CH3-CH3 interactions, IgG4 is able to engage in Fab arm exchange –4; altho–Fig. 1former 3–. Furtherformer 3–, 7 that dK of ∼10−10m (dK between 2 and 4 nm)  35, such dd 4 nm) 3–15, suggA). Moreover, besides structural variation between the IgG subclasses that potentially affects the stability, polymorphisms also result in structural variation within human IgG subclasses. Human IgG3 is particularly polymorphic, and a number of unique, serologically recognizable determinants  have been mapped to specific amino acid substitutions in the CH3 domains  , 16. HowFig. 2 domains –19. InteWe recently developed a method to monitor the kinetics of Fab arm exchange of IgG4 half-molecules in real time by monitoring the appearance of a Förster resonance energy transfer (FRET) signal upon recombination of IgG4 half-molecules containing two different fluorochromes (Fel d 1) were produced as described previously  and elution of the Fc constructs with 0.1 m glycine, pH 2.5–3. The eluate was neutralized immediately with 2 m Tris-HCl, pH 9, and dialyzed overnight to PBS. After dialysis, samples were stored at −20 °C. Concentrations were determined by A280 measurement using a Nanodrop ND1000.Fc fragments of different subclasses and allotypes were produced (CH2CH3 with an IgG4 hinge) by cloning synthetic constructs  into a pcDNA3.1 expression vector (Invitrogen) essentially as described before . Expresse.g. IGHG1*01 for IgG1, which corresponds to G1m(za). In this paper we examined several common IgG3 allelic structures: G3m(b*), G3m(c3c5*), and G3m(s*), where the shorthand nomenclature is: b* = b0, b1, b3, b4, b5, u, v; c3c5* = b0, b1, c3, u, c5; s* = b0, b3, b5, s, v  according to the instructions of the manufacturer. Unreacted dye was removed by repeated dilution/concentration using Amicon Centriprep centrifugal filter devices  until no dye could be detected anymore in the filtrate. The average degree of labeling was between 4 and 6 for IgG antibodies and ∼2 for Fc fragments.m dithiothreitol (DTT). Then the samples were incubated at the required temperature (10–37 °C), and the reaction was initiated by mixing equal volumes of both solutions into a thermostatted quartz cuvette. For IgG4-Fc-V397M/K392N, reactions were carried out at 10 μg/ml final concentration. B) 2 μg/ml IgG-488 and IgG-594 were mixed and equilibrated at 37 °C. Then the reaction was initiated by adding several microliters of a concentrated stock solution of GSH to a final concentration between 0.25 and 10 mm. Kinetics were monitored by measuring the appearance of a FRET signal using a Varian Cary Eclipse fluorescence spectrophotometer equipped with a thermostatted multi-cell holder . To compare experimental rate profiles, normalized fluorescence was calculated as F(norm.) = (tF − F0)/(Fmax − F0), where tF is fluorescence at time t, F0 is fluorescence at t = 0, and Fmax is the end-point fluorescence. The rate of IgG4 Fab arm exchange was also measured in a redox buffer that mimics plasma (m oxidized glutathione (GSSG), 3 μm reduced glutathione (GSH), 0.01 mm reduced cysteine (Cys), and 0.04 mm oxidized cysteine (CySS) in PBS. Activation parameters were obtained by measuring the dissociation rates at 4–5 different temperatures between 10 and 37 °C as described above; fitting a straight line to a plot of ln(kobsh/bTk) versus 1/T (Eyring plot) yields the activation enthalpy (slope = −R ΔH) and entropy (intercept = −R ΔS)  .m DTT in degassed PBS. Final concentrations of both IgG and IgG Fc were 20 μg/ml. The mixtures were incubated at 37 °C for 30 days (IgG1), 7 days (IgG2), and 24 h  and quenched by adding a 2-fold molar excess of iodoacetamide compared with DTT. Samples were stored at −20 °C until they were analyzed by applying 50 μl to a Superdex 200 HR 10/300 column (Amersham Biosciences), which was connected to a HPLC system (ÄKTAexplorer) from Amersham Biosciences. The column was equilibrated in PBS. Elution profiles were monitored by measuring UV absorption at 214 nm.IgG1, IgG2, IgG3, and IgG4 were mixed with the respective IgG Fc and incubated with 10 mm DTT (60 min/37 °C) and alkylated with 22 mm iodoacetamide. Serial 4-fold dilutions of reduced/alkylated IgG  were incubated with 0.05 ng/ml reduced/alkylated IgG2–488, 0.2 ng/ml IgG3, or 1 ng/ml IgG4/IgG1-K409R in PBS containing as a carrier protein 0.1 mg/ml certolizumab pegol, and 0.02% Tween 20 and incubated at 37 °C for up to 3 days before analysis. Between 50 and 1000 μl of a sample was applied using an autosampler to a Superdex 200 HR 10/300 column that was connected to an ÄKTAexplorer HPLC and eluted at 0.5 ml/min. Elution profiles were monitored by measuring the fluorescence (excitation/emission 488/525 nm) with a Prominence RF-20Axs in-line fluorescence detector . Dissociation constants were calculated as described before  to obtain a dose-response curve (2 + B2 + AB); therefore, the FRET signal is representative of the amount of dimers present in solution. The dissociation constant was calculated by fitting a homodimerization model to the data (see above).Equilibrium constants of G3*03 Fc, IgG4-V397M Fc, and IgG4-V397M/K392N Fc were determined by recording fluorescence spectra using either a Varian Cary Eclipse fluorescence spectrophotometer or a Nanodrop ND3300 of 2-fold dilutions of equimolar mixtures of Fc-488 and Fc-594 after incubation for 2 h at 37 °C in the presence of 3 mNon-reducing SDS-PAGE was performed according to the instructions of the manufacturer (Invitrogen) using 4–12% gradient gels. Samples were heated for 10 min at 70 °C. Iodoacetamide was added to the sample buffer to minimize artifacts . Gels weProtein three-dimensional structures were visualized using Discovery Studio 3.5, Accelrys.3DO3,3D6G,1OQO and 1OQX,3AVE) . If no interheavy chain disulfide bonds were present, the CH3-CH3 dissociation was the rate-determining step of the Fab arm exchange, allowing for direct quantification of the CH3-CH3 dissociation rate (C). To confirm that an exchange reaction was indeed taking place, antibodies were also incubated with Fc fragments of the respective subclass, and in all cases a 100-kDa structure corresponding to the product of the exchange reaction was observed (D). For IgG1, the dissociation rate was very low (kobs ≈ 5 × 10−7 s−1), an almost 5000-fold difference compared with IgG4 (kobs = 1.8 × 10−3 s−1). On the other hand, the dissociation rates for IgG2 and IgG3 were 60- and 4-fold lower compared with IgG4, respectively. It appears that structural variation among the subclasses other than Lys/Arg-409 influences the interaction strength between the CH3 domains. Moreover, the intrinsic half-life for dissociation of the CH3 domains was much shorter for all subclasses except IgG1, i.e. t½ = 16 days, 6.5 h, 28 min, and 6 min for the dissociation of IgG1, 2, 3, and 4, respectively, compared with their in vivo half-life of 21 days (7 days for IgG3). These results imply that the inter-heavy chain disulfide bonds of IgG2 and IgG3 will play a crucial role in determining their stability and raise the question if Fab arm exchange might be observed under conditions that mimic the in vivo redox conditions.We investigated the rate of dissociation of CH3 domains for all four human IgG subclasses using a previously developed FRET assay to monitor Fab arm exchange in real time B. If no ion rate . Removalion rate C. To conobserved D. For Igsupplemental Fig. S1), and all amino acid positions that differ between any two sequences are highlighted in a three-dimensional model of a CH3-CH3 dimer (A and B). Three of these reside at the CH3-CH3 interface: position 392 , position 397 , and position 409 , where it is Arg). In addition, amino acids at positions 356 and 358 (Asp and Leu for the G1m(a) allotype, and Glu and Met for the nG1m(a) isoallotype) might influence the CH3-CH3 interactions due to their close proximity to the CH3-CH3 interface. Based on these differences, we prepared a panel of Fc fragments that covers the variations in the CH3 interface between human IgG subclasses and allotypes. The panel includes four different allelic forms of IgG3 and several single-point mutants at positions 392, 397, and 409 . All Fc fragments contained an IgG4 core hinge, were fluorescently labeled, and were reduced by DTT to determine the dissociation rate at 37 °C using the FRET assay described above.Next, we systematically investigated the role of subclass-specific determinants and immunoglobulin polymorphisms that may influence the strength of the CH3-CH3 interactions. The sequence of the CH3 domains of all known polymorphic variants of human IgG subclasses were aligned , suggesting that this particular amino acid difference between IgG1 and IgG4 is the sole determinant that makes the CH3 interactions in IgG1 “IgG4-like.” It also implies that the structural variation at positions 356 and 358  does not result in an appreciable difference in stability.Introducing a K409R mutation in IgG1 (G1-K409R), a situation found in IgG4 , results in a structure with an increased exchange rate, that is equal to that of IgG4 C, suggesC). Conversely, no exchange was observed in this experiment for the G2-M397V mutant (kobs < 1 × 10−6 s−1). Thus, a methionine at position 397 significantly destabilizes the interactions between the CH3 domains, albeit to a smaller extent than an arginine at position 409.Comparing IgG1 and IgG2, the only structural difference in the CH3-CH3 interface is found at position 397. Indeed, the dissociation rate for the G1-V397M mutant was similar to IgG2 C. ConverC). However, G3*01 dissociates >10-fold faster compared with G3*06, demonstrating that an asparagine at position 392 results in weaker CH3-CH3 interactions leading to the relatively fast exchange reaction for this IgG3 allotype (C and 2C).The IgG3 used in Fig. 2allotype C and 2C.Interestingly, another IgG3 allotype, IGHG3*03 or G3m(c3c5*), resembles IgG4 in having an arginine at position 409 but also has the asparagine at position 392. In line with the abovementioned observations, G3*03 was found to dissociate ∼15-fold faster than IgG4, making it the least stable naturally occurring IgG3 allotype.i.e. Lys-392, Val-397, Lys-409, was as stable as IgG1, confirming the importance of these three key amino acids in explaining the differential stability of the CH3-CH3 interactions between IgG subclasses and allotypes thereof.Yet another IgG3 allotype, IGHG3*17 or G3m(s*), with identical residues as found in IgG1 at all the three positions differing between the subclasses in the CH3-CH3 interface, which were found here to influence the stability of the CH3-CH3 interactions, kobs = 1.6 × 10−1 s−1 was found nearly 6 orders of magnitude faster compared with IgG1 and >100 times faster than wild-type IgG4. In summary, we identified two new positions in the CH3 domains of IgG where amino acid variations as present among human IgG subclasses and IgG allotypes resulted in altered stability: Lys/Asn at position 392 and Val/Met at position 397.The combined effects of the destabilizing mutations at positions 392 and 397 in combination with Arg-409 in the backbone of IgG4 were also tested. Introducing a V397M mutation, found in IgG2 and some IgG3 allotypes, into IgG4 resulted in a labile structure that easily dissociates. Additionally, introducing a K392N mutation, found in some IgG3 allotypes, caused a further destabilization of the CH3-CH3 interactions, and a dK values for a number of the Fc constructs (A and B)  and a slope of 0.73. In other words, the variation in stability is dominated by variation in the dissociation rate and much less by variation in the association rate. For IgG1 no dissociation constant could be measured. However, a reasonable estimate was obtained by extrapolating the linear trend between dK and kobs; the dissociation rate measured for IgG1 translates to a dK between 10−13 and 10−14 M, which implies that the interdomain interactions between the CH3 domains are exceptionally strong for IgG1.To learn more about the CH3-CH3 interactions, we also measured the nstructs . For thenstructs . For thenstructs , A and BA and B) . The resG, ΔH, ΔS). Although the dissociation rates suggest that the contributions of mutations at these three positions are essentially additive, activation parameters demonstrate substantial non-additive contributions in the enthalpy and entropy of activation (versus G3*01) or in IgG4 (IgG4 versus G4-K392N). The interactions of these amino acids apparently mutually affect each other. The enthalpic contributions dominate the dissociation process and, therefore, probably also the interaction strength.Additional insight about the effects of the different mutations can be obtained from activation parameters , it suggests the hinge disulfide bonds are even more important for maintaining structural integrity of antibody molecules than previously recognized, and even transient reduction may result in Fab arm exchange. Therefore, we also investigated the susceptibility toward Fab arm exchange for different hinge configurations at different redox conditions. Two “IgG1-like” hinge configurations were examined: a “hinge-stabilized” version of IgG4 bearing an S228P mutation (a mutation that is present in a number of therapeutic IgG4 antibodies currently in development) as well as IgG1-K409R and two IgG4-like structures, IgG4, and IgG1-K409R/P228S, abbreviated as IgG1-KRPS. The latter IgG1 mutant contains both features that are unique to IgG4 and allow facile Fab arm exchange.m, the rate is only a factor of 5–10 lower compared with 10 mm GSH. On the other hand, the IgG1-like hinges do not participate in observable Fab arm exchange at 0.25 mm GSH, and a slow Fab arm exchange becomes noticeable only at concentrations of GSH between 1 and 5 mm. This much steeper dependence on the GSH concentration reflects a different mechanism of Fab arm exchange (see “Discussion”). Neither IgG1-K409R or IgG4-S228P was found to exchange in vivo in animal models   for wt IgG4, its S228P mutant, and for both IgG1 mutants . Howeverl models , 58. Thefor IgG4 , 16. Nev IgG4 , it was especially relevant to examine the susceptibility for Fab arm exchange at mildly reducing conditions. At 0.25 or 1 mm GSH no Fab arm exchange was observed, which suggests that the propensity to participate in Fab arm exchange in vivo is probably low (B and D). However, at 5 mm GSH Fab arm exchange was efficient, and the approximate rate of exchange was ∼3-fold lower compared with the situation in which the disulfide bonds were fully reduced by DTT. The steep concentration dependence suggests a cooperative reduction of disulfide bonds.For IgG3, with its extended and multiple disulfide-linked hinge C, it wasably low , B and DE). The variation of the rate of exchange with concentration of GSH is reflected in the appearance of noncovalent isomers, demonstrating that breaking of disulfide bonds, or in the case of IgG4, the formation of intrachain hinge isomers controls if, and how fast Fab arm exchange may take place.The formation of noncovalent hinge isomers was also examined at different concentrations of GSH for all subclasses by SDS-PAGE E. The vaA). In IgG1 and IgG4, Cα-Cα atom distances in the vicinity of Val-397 are essentially identical, except for those from the DE loop, which are affected by Lys/Arg-409 variation . The Leu-398–Lys-392′ and Asp-399–Lys-392′ Cα-Cα distances are not affected by the V397M substitution. The effects of Met-397 on the CH3-CH3 interface are thus local in nature, and the average buried surface area for the IgG2-Fc CH3-CH3 interface (2319 Å2) is comparable with the IgG1 average (2313 Å2).For IgG4 it has been described that Arg-409 disrupts a network of water-mediated hydrogen bonding that is conserved in IgG1, which explains the weaker CH3-CH3 interactions , 55. HerFig. 6ariation , 55. In B). It was found that some Asn-392 conformers could form hydrogen bonds with the Asp-399′ side chain and Leu-398′ main chain as well as surface water molecules. However, other conformers clashed with Phe-405′ and Asp-399′ side chains. Additionally, one Asn-392 conformer could disrupt a conserved water molecule network at the CH3-CH3 interface (W1–4) in a similar manner to Arg-409 in IgG4  acts to counterbalance the overall instability. These results highlight the importance of the hinge disulfide bonds in maintaining the structural integrity of IgG2 and IgG3 molecules. Furthermore, these results imply that for any therapeutic antibody, but in particular IgG4 antibodies with a stabilized hinge (S228P) and bispecific antibodies, it is important to ensure proper formation of hinge disulfide bonds. This is not necessarily the case  gene. Here we demonstrate that these polymorphisms lead to variations in the strength of inter-heavy chain interactions in IgG3 by approximately 6 orders of magnitude. Interestingly, one strategy to enhance the potency of therapeutic antibodies for (tumor) cytotoxicity involves the introduction of IgG3 features into an IgG1 backbone or the use of complete IgG3 allotypic variants with extended half-life . Natsumeet al. , 63 repoet al. , 63, weret al.  Asn-392 conformations would cause steric clashes or even disrupt the conserved water molecule network. From a recent study it is known that Arg-409 adopts two conformations at the IgG4 CH3-CH3 interface, one of which is compatible with a conserved water molecule network, whereas the other is not, resulting in reduced hydrogen bonding and a lowering of the buried surface area . PerhapsSequence variation at position 397 is not unique to human IgG subclasses. For example, residue 397 is valine in murine IgG2a and -b but isoleucine in IgG1 and IgG3. Analysis of the available crystal structures of murine IgG1, IgG2a, and IgG2b , 66–68 d66–k ∼ [GSH]2, because two disulfide bonds need to be broken simultaneously before dissociation is possible; for IgG4 the rate changes ∼4-fold upon going from 0.5 to 5 mm GSH, reflecting that the major non-covalent isomer being formed is the intrachain form . The steep concentration dependence for stabilized hinge antibodies explains why on the one hand in vivo Fab arm exchange is not observed anymore  .At mildly reducing conditions, the rate of Fab arm exchange of IgG4 with a “stabilized” hinge (IgG4-S228P) depends markedly differently on the GSH concentration in comparison to wild-type IgG4; for IgG4-S228P the dependence is approximately  anymore , 58, wheobodies) . The milin vivo life-span; for IgG2 and IgG3 the interheavy chain disulfide bonds are essential to prevent half-molecule dissociation, whereas for IgG4 the labile hinge disulfide bonds favor half-molecule exchange.In summary, we investigated the dynamics of inter-heavy chain interactions of human subclasses and allotypes using real-time monitoring of Fab arm exchange. We identified structural variation between human IgG subclasses and allotypes at three amino acid positions  to alter the interdomain interactions of CH3 by >6 orders of magnitude. These interactions are only sufficiently strong for IgG1 to maintain the structural integrity of the molecule during its


More than 60% of the construction workers had a negative attitude toward HIV/AIDS-infected individuals. The source of workers’ knowledge toward HIV/AIDS mainly came from TV (35.8%), newspaper (14.3%), family and friend (13.1%) and others (28.2%). Chinese migrant workers in general lack knowledge about HIV/AIDS. Our study suggests prevention programs should be encouraged and these may have the potential role to limit the emergence of China’s HIV/AIDS epidemic.The objective of the study was to describe HIV/AIDS knowledge, attitudes, risk behaviors, and sources of information among construction workers in China. A cross-sectional survey of 458 construction workers was conducted among 4 construction sites in Shenyang city in 2006. All 458 participants were individually interviewed in a private setting by a trained team of medical researchers using a structured questionnaire, which included questions on general personal information and the knowledge, attitudes, practice questions and the favorable mode of health education. A total of 428 valid questionnaires were collected. Data entry and statistical analyses were performed using the SPSS 13.0. Our results indicated that the majority of construction workers in China are sexually active youths and adults with limited education and poor knowledge of HIV/AIDS. The proportions of correct answers to questions about HIV/AIDS ranged from 4.9% to 70.7%. The score was significantly different by education level (χ China’s HIV/AIDS epidemic has gone through two stages . It is now in the third stage - ‘expansion’. The UNAIDS estimates that 10 million Chinese may be infected with HIV/AIDS by 2010 . Moreover, additional content was added to the questionnaire based on other relevant previous studies.We used a self-administered HIV/AIDS questionnaire written in Chinese, the content of which was initially drafted following by a previous study conducted in China . Percentage distributions of knowledge, attitude, and practice of HIV/AIDS were tabulated and χShenyang City is the capital of Liaoning Province, located in the north of China. It has a population of 7.2 million in 2006. We selected randomly 4 construction sites for the investigation from Shenyang City. In this study, construction workers were individually interviewed in a private setting by a team of trained medical researchers using a self-developed questionnaire, which included general personal information and the knowledge, attitude, practice questions and the favorable mode of health education. Out of all 458 construction workers, 428 completed their questionnaires, representing an overall response rate of 93.4%.All 428 construction workers between the age of 18 and 52 years old in those sections were invited to participate in the survey. All participants are male. This studied population is 72.4% married, 26.1% single, and 15% divorced. Their educational level are varied: illiteracy (10.9%), elementary school (28.1%), junior high school (54.8%), senior high school and upwards (6.2%). These workers mainly come from Sichuan province (26.2%), Liaoning province (14.1%), Henan province (14.5%), Hubei province (11.1%), and other provinces (34.1%).p<0.01), while the Kaiser–Meyer–Olkin measure of sampling adequacy was 0.72, justifying the application of the factor-analytic procedure.The pilot-investigation of the 50 construction workers had a Cronbach’s alpha coefficient of 0.78. An exploratory factor analysis (EFA) was first applied to ensure the structural validity. The analysis was performed using principal axis factoring with varimax rotation on the correlations of the observed variables. The Bartlett’s test of sphericity was found to be significant .Table The results show that people had prejudiced attitudes towardsAIDS patients and can’t accept living with AIDS patients. 82% of construction workers thought that people with HIVshould be isolated; 62.6% of subjects were not willing to have meals with people who have HIV; but fortunately, 87.1% of respondents considered that people with HIV should be helped more; and 73.6% of subjects were willing to take care of families of patients with HIV.Table Our study shows that the source of information about AIDS includes TV (35.8%), newspaper (14.3%), family and friend (13.1%), and radio (4.2%). A total of 82.2% of construction workers wanted to acquire more HIV/AIDS knowledge. The favorite modes of learning are “Doctor’s lectures or consultation toward AIDS” (28.3%), “providing booklet” (16.6%), “looking on CD” (14.7%).The construction workers we studied are mostly sexually-active youth and adults from rural areas with low education level and low income. Our study shows the HIV/AIDS knowledge among these workers is related to the level of education. The lower the education level, the less knowledge the construction workers have about HIV/AIDS. It will be necessary to improve the education level of rural people. Because of these workers’ low-income, an orderly medical physical examination is not feasible. In addition to discrimination against HIV/AIDS patients, they weren’t expected to be HIV tested, which increases the possibility of unknown spread of HIV.Our investigation revealed that the majority of construction workers in Shenyang were migrants from Sichuan province (26.2%) and Henan province (14.5%). This segment of construction workers may play an important role in spreading the disease. We must pay attention to these people’s HIV testing and education to decrease the lack of awareness regarding HIV prevention. Still, the main transmission route of HIV is through sexual contact, rather than blood transfusion and drug-use.Our study shows that construction workers have little knowledge about HIV risk behaviors, which makes them at greater risk of contracting HIV. In promoting and disseminating HIV/AIDS knowledge, we can select TV, newspaper, and family-friend interchanges. Most of construction workers (82.2%) wanted to know more about HIV/AIDS and 60.2% wanted to take part in a free course of lectures. So we can make use of these ways to propagate the HIV/AIDS knowledge.There are some limitations in this research. The study was limited to four building sites. Another limitation is that the study doesn’t show any information about any risky sexual behaviors, therefore can not determine the risk levels for this group. Because of the limited conditions, we can’t investigate the construction workers’ HIV infection rate, which is estimated to be higher than the rate among other population groups. Finally, only 428 construction workers were included in our study; future studies with bigger sample size are needed.Based on our findings, we suggest: (a) we must pay more attention to construction workers in China in order to prevent the HIV/AIDS epidemic from spreading quickly; (b) education programs are in urgent need for this group of people and such programs should aim at both equipping these individuals with necessary knowledge about HIV/AIDS and correcting misunderstandings; (c) HIV/AIDS interventions should incorporate programs that can help construction workers improve their income level because they have a strong desire in this regard; (d) a free course of HIV/AIDS lectures should be regarded as a useful tool in planning interventions.We found that construction workers mostly came from rural areas, had low education levels, were sexually active, and had low incomes. They have little knowledge about HIV/AIDS and related risk behaviors. China should implement HIVAIDS prevention programs for this population group.


There is now strong evidence of the presence of a vast integrated neural system, which involves the hippocampus and several parahippocampal regions, dedicated to spatial navigation and memory. 'Place cells' and 'Grid cells' have been identified as neural correlates of the ability to remember an environment and to successfully move through it.The storage of a spatial map in the hippocampal circuits poses important computational issues. It asks for an extension of the classical notion of attractor, successfully used to model the storage of discrete memory items, like episodic memories, to a new entity that captures the continuous nature of space.In a continuous attractor activity smoothly changes with the position of the animal in the environment, so that each point in space is uniquely represented in the hippocampus. This situation corresponds to the presence of an infinite number of distinct stable configurations of the system, each for a different point of the environment.It is not known how much this idealized notion corresponds to the actual structure of the maps stored in the hippocampus, and how well attractor dynamics can approximate a continuous representation.We address these questions, within a simplified mathematical network model. The model network simulates the storage in CA3 of a spatial representation, its retrieval and its transfer to CA1. Through both analytical calculations and computer simulations we evaluate the ability of the network to represent different points in space and we study the properties of the attractor landscape when different kinds of disorder  are introduced in the system. Moreover we investigate the possible role of CA1 in the realization of a continuous attractor.We find that even networks of considerable size can only approximate the idealized notion of a 2D quasi-continuous dynamical attractor, but that the presence of CA1 generates a 'smoother' representation of space.


IgG4-related disease (IgG4-RD) is a multiorgan inflammatory disease in which diverse organ manifestations are linked by common histopathological and immunohistochemical features. Prospective studies of IgG4-RD patients are required to clarify the natural history, long-term prognosis, and treatment approaches in this recently recognized condition. Patients with IgG4-RD have different organ manifestations and are followed by multiple specialties. Divergent approaches to the assessment of patients can complicate the interpretation of studies, emphasizing the critical need for validated outcome measures, particularly assessments of disease activity and response to treatment. We developed a prototype IgG4-RD Responder Index (IgG4-RD RI) based on the approach used in the development of the Birmingham Vasculitis Activity Score for Wegener's granulomatosis (BVAS/WG). The IgG4-RD RI was refined by members of the International IgG4-RD Symposium Organizing Committee in a paper case exercise. The revised instrument was applied retrospectively to fifteen IgG4-RD patients at our institution. Those scores were compared to physician's global assessment scale for the same visits. This paper describes the philosophy and goals of the IgG4-RD RI, the steps in the development of this instrument to date, and future plans for validation of this instrument as an outcome measure. Measurement of disease activity is critical for longitudinal assessments in both observational studies and clinical trials. In the field of rheumatology, more than 250 assessment tools have been developed and validated to evaluate pathology, symptoms, function, and health status of patients with rheumatic diseases . Such inIgG4-related disease (IgG4-RD) is an increasingly recognized immune-mediated disease that is characterized by a lymphoplasmacytic infiltrate enriched with IgG4-positive plasma cells and a distinctive storiform fibrosis of affected organs . CommonlA disease responder index is a tool designed to detect any changes in disease activity and identify improvement and worsening in the same and/or different organ systems. A responder index permits objective quantification of the treatment response by providing standardized outcome measures. Assessing clinical response and not simply serologic response is increasingly important to establish endpoints in randomized control trials.No randomized control trials have been conducted for IgG4-RD treatment to date . ManagemTwo major features of IgG4-RD pose significant challenges for the development of outcome measures. The first is the complex, multiorgan system nature of this disease, which makes it difficult to summarize the state of disease activity across all organs. The second is the fact that the stage of disease activity can differ across organs, such that a patient can have active inflammation likely to respond to immunosuppression in one organ and advanced fibrosis (less likely to respond to treatment) in another.We have developed an IgG4-RD responder index (IgG4-RD RI) for use as an outcome measure in an ongoing pilot trial of rituximab in this condition. We intend that this instrument will measure not only disease activity but will also incorporate features that capture the need for urgent treatment and catalogue disease-related damage. This paper is designed to provide information on the development and implementation of the IgG4-RD RI. We report the philosophy behind the development of the IgG4-RD RI to date, the steps taken to create the instrument through the enlistment of assistance of international experts in this condition, and the plans for completion of the IgG4-RD RI validation process.http://www2.massgeneral.org/pathology/symposium/IgG4_related_systemic_dis.asp]. This group was comprised of 39 experts from 9 countries, with subspecialty expertise in rheumatology, gastroenterology, allergy/immunology, nephrology, surgery, pathology, and radiology. Further revisions were made following a simulation exercise involving six paper case descriptions of real patients, completed by IgG4-RD symposium participants. Finally, both the IgG4-RD RI that emerged from these development steps and a physician global assessment (PGA) were used to assess the disease retrospectively in terms of disease activity and damage. The Pearson's correlation coefficient was then calculated to compare the IgG4-RD RI and PGA responses.The IgG4-RD RI was designed to assess disease activity from visit to visit using clinician-generated assessments of both objective and subjective measures. The IgG4-RD RI uses a scoring system from 0–4 for each organ system or site and asks the clinician to rate the extent of disease activity and damage at the time of the clinical encounter. The IgG4-RD RI was revised by the Organizing Committee of the International IgG4-related Disease Symposium, held in Boston in October, 2011 . For. For3]. Lists of the most common symptoms and signs within a given organ system are included in the IgG4-RD RI instructions (see the appendix), principally as a reminder to the clinician of the possible disease manifestations to consider when scoring disease activity and damage. The physician simply denotes on the form the presence or absence of symptoms for a given IgG4-RD site. This feature of the instrument ensures that a subjective measure is included. Good clinical judgment and a thorough knowledge of the disease manifestations of this condition are essential, as with any clinical responder index.Capturing the need for urgent treatment is an important aspect of the IgG4-RD RI. Some disease manifestations of IgG4-RD require the immediate institution of treatment to prevent permanent organ damage. For example, IgG4-related sclerosing cholangitis can lead to cirrhosis within several months of diagnosis and requires the prompt initiation of therapy. In contrast, the lymphadenopathy of IgG4-RD remains unchanged for prolonged periods in many patients and may never require treatment. The “urgent” column in the IgG4-RD RI is designed to capture aspects of the disease that require the immediate start of immunosuppression in order to preserve organ function.The score for an organ site is doubled when the need to initiate treatment for active IgG4-RD at a particular or organ/site is considered urgent. For example, if a patient has new IgG4-related sclerosing cholangitis, the total score for that organ/site would be 6 instead of 3. Similarly, if the patient's biliary status has worsened despite therapy since the time of the last visit and an urgent escalation of therapy is required to treat the IgG4-related sclerosing cholangitis, then that organ score would be 8 rather than 4. Only the score of the individual organ site is doubled in this setting, not the total IgG4-RD RI score.The concept of damage is related directly to that of disease activity. Consequently, the two concepts must be considered in tandem. Organ damage results from active disease and in some cases both active disease and damage can be present in the same organ system simultaneously. In other cases, organ dysfunction is related more to damage than to active IgG4-RD. Immunosuppression must be targeted to active IgG4-RD, not to damage resulting from previously active therapy. The most appropriate use of immunosuppression is to control active disease and prevent disease-related damage. It is particularly ideal to employ immunosuppression at a stage of disease when the histopathology is characterized by a lymphoplasmacytic infiltrate rather than a predominance of acellular fibrosis .The clinical assessment of damage can be challenging. Radiographic studies such as computed tomography (CT) and positron emission tomography with CT (PET-CT) can aid the clinician in determining which organs have been damaged. For example, even conceding that active disease might be present simultaneously with damage within the pancreas, the finding of atrophic changes by CT scan within that organ would be considered to be the result of damage. In such a case, both active disease and disease-related damage should be scored.Scoring also includes a consideration of the serum concentration of IgG4. The serum IgG4 level may become elevated in a patient experiencing an active flare , 10. HowThe sum of the disease activity in all of the organ sites plus the serum IgG4 concentration score  yields the total activity score. An individual active organ site is doubled for urgency and added to the other organ sites. This number can be compared between visits to assess the disease activity over time as well as being used for a clinical trial endpoint. The longitudinal recording of damage, though not included in the overall disease activity score, is essential to the formulation of the patient's overall outcome.Glucocorticoids are the cornerstone of IgG4-RD treatment, and most patients respond promptly to this treatment, at least initially. Thus, careful recording of the dose of prednisone  in the interval between the current visit and the preceding one is essential to a full understanding of the degree of disease activity.International IgG4-RD Symposium . Participants were asked to score the simulation exercises using the IgG4-RD RI. The participants received written instructions on how to apply the IgG4-RD RI but did not attend a training session (the appendix). Twenty-one individuals participated in this exercise, providing valuable feedback from a cross-section of investigators interested in IgG4-RD. The physicians who completed the exercises included a variety of subspecialists, particularly rheumatologists and pathologists  rather than resolved . Another scoring discrepancy resulted from incorrectly scoring patients off treatment who were recurrent (“3”) as if they were receiving treatment (“4”). A third error was failing to double the organ/site score, when disease requiring treatment urgently was present. Comments from the participants in this exercise contributed substantially to important revisions of the draft instrument. We reformatted the scoring sheet in order to address the common scoring differences from the simulation case exercises.P < 0.0001).The next step in the development of the IgG4-RD was the retrospective use of the instrument for fifteen individual clinic and in-patient evaluations among patients in the Massachusetts General Hospital IgG4-RD Registry. Two blinded rheumatology experts scored an IgG4-RD patient visit using either the IgG4-RD RI or the physician's global assessment scale (PGA). These results were then compared . The PeaAs the field of IgG4-RD is poised to move beyond the descriptive phase of the disease, validated outcome measures are required to advance the understanding of this condition and the assessment of new treatment approaches. The current iteration of the IgG4-RD RI marks an important step toward the availability of useful outcome measures in this disease. We anticipate that additional validation steps for this instrument will be required, but this paper describes accurately the philosophy and goals behind the IgG4-RD RI. Its methods and appendices will serve as important guidance documents in the future.The development efforts to date have created a one-page instrument supported by the instruction manual shown in the appendix. Data included on this single page include indications of disease activity across a full spectrum of potential organ involvement; the serum IgG4 concentration; assessments of the need for treatment on an urgent basis; the recording of damage in organ systems; the sum of recent glucocorticoid use. Expertise with the use of the IgG4-RD RI may, therefore, become a concise and important tool for clinical trials and other investigations related to this disorder.Although the developers of the IgG4-RD RI have relied significantly upon the BVAS/WG in creating this instrument, the IgG4-RD RI differs in important ways from the BVAS/WG. The “urgent” column in the IgG4-RD RI highlights features of the disease that require the prompt institution of treatment and is, therefore, analogous to the “major” designations given to some organ system manifestations in the BVAS/WG . HoweverThe IgG4-RD RI will find its greatest use in the research setting, either in the context of clinical trials or in other types of investigations that require the careful longitudinal assessments of patients' clinical status. Because consistency of its application from visit to visit is critical, it will be most useful to ensure whenever possible that the same investigators complete the IgG4-RD RI for the same patient across all visits.Significant debate now exists within the community of IgG4-RD investigators about the utility of serum IgG4 concentration measurements in the diagnosis and management of this disorder . InclusiThe simulation case exercises illustrated some shortcomings in early iterations of the IgG4-RD RI that led to appropriate revisions of the original index. The experience with the simulation exercise highlighted the importance of adequate training with the instrument prior to its use in the research setting. The IgG4-RD RI is simpler than many clinical assessment tools for multiorgan diseases, but both a thorough understanding of the clinical breadth of IgG4-RD itself and a high degree of familiarity with the index are required in order to employ it effectively. We anticipate that a focused period of instruction for investigators in the context of a formal training course will be required before this tool can be used in the context of a clinical trial.In conclusion, progress in IgG4-RD will be contingent upon the ability to assess patients rigorously in a longitudinal manner, using validated outcome measures. The IgG4-RD RI described in this paper represents a broad effort at the development of a disease activity and responder index that can be employed in clinical trials and other investigations of patients with this emerging immune-mediated condition. The next steps in validation will include a multicenter study of patients recruited from a core group of sites with extensive experience in the diagnosis and management of this disease.The supplementary appendix includes the instructions for use of the IgG4-related disease responder index (IgG4-RD RI). This instruction manual describes how both disease activity and damage are recorded on the IgG4-RD RI scoring sheet . Scoring rules are described for the organ/site scores as well as serum IgG4 concentration and total scores. The definition of a damaged organ is provided and how to record damage. Finally, a version of the IgG4-RD RI that includes all of the organ/site common disease manifestions is included.Click here for additional data file.


Greater Mekong Sub-region (GMS) is composing of the border of Thailand-Myanmar, Republic Lao, and China with various of geographic and cultures and more than 200 million people live there. The migration of population is simple scenario at these areas. Last few decades Thailand has been reported of the highest prevalent of HIV/AIDS particularly in the northern of Thailand. The study aimed to investigate the effect of HIV/AIDS at the areas of GMS.This retrospective cohort study design aimed to investigates the HIV situation among the people who immigrated into Thailand in GMS. The systemic data extraction from the medical records from 19 hospitals which located in the border of Thailand during 1990-2009 was analyzed. The instruments had been detected in validity and reliability. Chi-square test was used for identifying the statistical significant at the alpha=0.050.Totally 1,303 cases had been detected. Of 84.50% were still alive, 57.27%were male. Myanmar were 56.52%, 26.44% were unknown, and 11.10%were Republic Lao, 5.24%were Chinese, and 1.00% was Cambodian. Of 37.53% were aged 21-30 years old, 33.15% were aged 31-40 years old, and 12.89% were aged 41-50 years old. Of 38.94%were agricultural, 34.56% were employee, 4.15%were student, and 3.46% were young children. Of 51.65% were full bone AIDS, and 28.32 were symptomatic AIDS. Of 92.31% had infected by sexual intercourse, 6.06% were mother to child, and 1.63%wereIDU. Distribution of age by sex was statistical difference . Being female had a longer live than male . The live status was different according to nationality  and risk factor .The cooperation between countries to control the HIV spreading is immediately need for the GMS region especially the free trade market in 2015.