Objective. Immunoglobulin-G4-(IgG4-) related disease (IgG4 RD) is a fibrosing process characterized by a significant infiltration of IgG4-secreting plasma cells. IgG4 RD can affect almost all organs including salivary glands. Whether IgG4 RD plays a role in the development of sicca syndrome and particularly dry mouth syndrome remains to be investigated. Methods. We conducted a monocentric cohort study for two years to search for IgG4 RD features in patients with dry mouth syndrome using immunostainings of labial salivary gland specimens with anti-IgG4 antibody. Results. Among 60 patients presenting with dry mouth syndrome who underwent labial salivary gland biopsy, 18 showed positive immunostaining with the anti-IgG4 antibody including 4 patients with typical systemic IgG4 RD. Five also fulfilled criteria for Sjögren's syndrome. Conclusion. These findings suggest that clinical forms of IgG4 RD salivary involvement without salivary swelling may occur. This salivary involvement is probably overlooked in everyday practice and could represent a mild form of IgG4 RD.  The symptoms of dry mouth syndrome frequently cause patients to consult with physicians. More than 10% of the population reports symptoms linked to oral and/or ocular dryness. The causes of xerostomia are numerous including Kuttner's tumor (KT) and Mikulicz's disease (MD). Both are mainly characterized by enlargement increase in the volume of salivary glands, with submandibular glands involvement in KT and parotid glands in MD associated with lacrimal induration. Until 2005, MD and KT were classified as subtypes of Sjögren's syndrome (SS) .The concept of IgG4 RD was suggested in 2003 by Kamisawa et al. .However, very little data are available in Western countries and the prevalence, characteristics of the salivary involvement of IgG4 RD, and its relationship to SS have yet to be defined.The aim of this study was to determine if IgG4 RD features were present in French patients who had dry mouth syndrome whether or not they have SS. A cohort study was conducted retrospectively in a French university hospital for two years between January 2009 and December 2010. Thus, we reviewed all the medical charts of adult outpatients and patients who were hospitalized and had a labial salivary gland biopsy (LSGB) between January 2009 and December 2010, for any reason whatsoever. Patients with a context of sarcoidosis, amyloidosis, lymphoma, salivary lithiasis, drugs likely to induce a dry mouth syndrome, which had histories of cervical irradiation or systemic sclerosis, hepatitis C, or HIV infection were excluded. Then, immunostaining with anti-IgG and anti-IgG4 antibodies of the biopsy samples was performed for patients who presented with dry mouth syndrome according to the criteria in Box  The LSGB samples were assessed in one of the two histopathology laboratories of this university hospital depending on which department the patient required. The LSGB samples were fixed in alcohol-formalin-acetic acid, dehydrated, embedded in paraffin, and sliced into six 5-micron-thick sections. An ultraView kit  was used to perform immunostaining with the sheep anti-IgG antibody IgG (Gamma-heavy chain) antibody-1  and the murine antihuman IgG4 antibody clone HP 6025 . Diaminobenzidine was used as the reagent to visualize the antigenic sites. The immunostaining was performed in a single pathology laboratory. The IgG4/IgG tissue ratio was considered positive if the ratio was greater than or equal to 40%. Biopsies of other organs were examined by the same technique.All included patients had positive Schirmer test and 47 of them were tested for autoantibodies to extractable nuclear antigen (ENAs). Those who were not tested for ENAs had a focus score equal to 1 or more on the LSGB. Measurement of saliva production had been performed by sugar test. A dry mouth syndrome was defined by not totally dissolved sugar after 3 minutes.Using these data, the patients were divided into 4 subgroups according to the characteristics of their sicca syndrome: those who fulfilled SS criteria of the American-European Consensus Group  only , biological criteria  by IFI on HEP2 cells, ENAs including anti-SSA and anti-SSB on EliA Symphony Well Phadia , rheumatoid factor (rf), and thyroid-stimulating hormone (TSH)), histopathological criteria , and a therapeutic criterion .We performed, with STATAv11 , ANOVA test followed by a posthoc Tukey-Kramer test for continuous variables and Fisher's exact test followed by a Marascuilo procedure for categorical variables.Ninety-nine patients had an LSGB between January 2009 and December 2010. Among these 99 patients, 60 presented with subjective sicca syndrome according to the Vitali criteria [n = 2) or visible lymphoplasmacytic infiltrates were absent (n = 3). The immunostaining could not be performed in 5 cases because the amount of the residual biopsy material was insufficient (n = 17) or on another tissue (n = 1) ; group B, 66 ± 10 years (54–77); group C, 58 ± 7 years (47–73); group D, and 66 ± 14 years (38–89). There was no significant difference in the age between patients of groups A, B, C, and D. Of the patients who suffered from sicca syndrome, 30% presented with salivary involvement due to IgG4 RD with or without SS, 36.7% fulfilled only the diagnostic criteria of SS, and 33.3% did not fulfill the criteria for either pathology.There were 49 women and 11 men with an average sex ratio (M/F) of 1/4.5. The highest proportion of women (1/5.5) was observed in group C.n = 4) occurred mainly in the patients from group C (n = 3), but the difference was not statistically significant. Lastly, extra-salivary involvement was observed in 46.6% of the cases (n = 28). An atopic background was observed in 18 patients including 8 in group A. Swelling of the main salivary glands (n = 13), central or peripheral nerve involvements (n = 5), haematological involvements (n = 3) including one patient with pernicious anaemia and vitiligo (n = 1), primary biliary cirrhosis (n = 2), fibrosing hepatitis (n = 1), interstitial pulmonary involvements (n = 2), thyroiditis (n = 1), dacryoadenitis (n = 1), skin involvements (n = 2 including livedo (n = 1), and urticaria (n = 1)), cervical lymphadenopathy (n = 1) and tubulointerstitial involvements of the kidneys (n = 2).Among the patients with extra-salivary involvement, some had arthritis .Two of the patients presented an extra-salivary involvement due to IgG4 RD that was proven histologically (liver n = 2) or associated with SS criteria (n = 2). The highest IgG4 serum level (26.1 g/L) was obtained in a patient with a systemic form of IgG4 RD. IgG4 serum levels tend to be higher in group B than in group A (P = 0.056). The serum levels of the other IgG subclasses  obtained in 7 patients were normal.Due to the study design, the IgG4 serum level was measured in only 13 patients with a level greater than 1.35 g/L in 5 patients. Among these 5 patients, 4 showed characteristics of IgG4 salivary involvement only (n = 11). ENAs positivity was significantly higher in group A than in group C (P < 0.0001) and tend to be more frequent in group A than in group D (P = 0.051). TSH measurement was performed in 26 patients and was elevated in 3, each of whom belonged to a different group .Autoimmune features (ANA and RF) were present in many patients, with ANA levels greater than or equal to 1/160 in 24 patients of the 54 who were tested and positive RF in 7 of the 28 patients. ANA levels (>1/160) were observed in 62% and 25% of tested patients in groups A and C, respectively, and in 60% of the patients in group B. The anti-ENA antibodies test was positive in 14 of 49 tested patients and most patients who tested positive belonged to group A (n = 26) presented with SS criteria alone or associated with a positive IgG4 salivary immunostaining. A focus score equal to 1 or more was more frequently found in group A than in group C (P = 0.0058) and group D (P < 0.0001). A focus score equal to 1 or more was more frequent in group B than in group C (P = 0.0001) and group D (P < 0.0001). These expected results confirmed that a focus score equal to 1 or more was strongly associated with a diagnosis of SS, with or without features of IgG4 salivary involvement.A focus score equal to or greater than 1 was observed in 34 patients. Of these patients, 76%   showed positive immunostaining on LSGB more frequently than those from group A and D (group A versus B: n = 11). Paradoxically, this improvement was not more marked in patients with stigmata of IgG4 salivary involvement. Corticosteroids, given at the physician's discretion after the biopsy procedure, were used in 20 patients. No objective evaluation of the clinical response to steroid was performed. However, a subjective response in sicca syndrome was observed in 55% of cases , SS (n = 8), or lymphoepithelial sialadenitis, which is a benign pathological form of sialadenitis linked to SS in 40% of cases (n = 4),in 2010, Geyer et al.  comparedlastly, in 2009 and then in 2010, Masaki et al. compared 64 cases of MD in Japanese subjects with 31 cases of SS , 13.Three major studies have tried to address the clinical and biological differences between SS and MD or KT:The data from these studies showed that sialadenitis linked to IgG4 occurred more frequently in women but with a sex ratio (M/F) that was proportionally higher in men than in SS. Indeed, the sex ratio (M/F) in IgG4 sialadenitis in Asian patients appears to vary between 1/2  and 1/3.The average age at onset of sialadenitis linked to IgG4 varies between 61  and 66.7One group of our patients, even though small, fulfilled the SS criteria and showed IgG4-positive immunostaining. These findings suggest an eventual coexistence of SS and IgG4 salivary involvement features in patients with sicca syndrome. However, a focus score ≥ 1 was found in our patients with the expected frequency reported in the literature . This obThe relationships between IgG4 and allergic disorders were described in 1974, fuelling the debate on whether the origin of IgG4 RD was autoimmune or allergic . In our A serum IgG4 level greater than 1.35 g/L was detected significantly more frequently in the group with SS associated with IgG4 salivary involvement features than in the group with SS alone. Regardless of the affected organ, the IgG4 serum level is often significantly higher in IgG4 RD than in other causes . These dAutoimmune biological features are usually observed in IgG4 RD patients. In salivary involvement, the presence of ANA and positive RF was frequent. The presence of ANA occurred in approximately 28% of cases in Yamamoto's study . HoweverFrom a pathological point of view, IgG4 RD is characterized by the association of a lymphoplasmacytic infiltrate rich in IgG4+ plasmacytes, a fibrosis, and in situ thrombosis in variable proportions . FibrosiFrom a therapeutic point of view, the steroid responsiveness of IgG4 salivary involvement is well known . MD or KTo the best of our knowledge, our study included the largest cohort of patients of Western origin with IgG4 salivary involvement features. The findings highlight the possible association between IgG4 salivary involvement features and SS, which leads us to believe that an overlap of these two pathologies exists. Furthermore, the clinical characteristics of the patients in our study show probable phenotypic differences in IgG4 salivary involvement between the Asian and Western patients. There are some limitations in our study. First, the control group comprises patients that are “nonhealthy” because of sicca syndrome and the 4 groups are not matched. Second, as in all retrospective studies, our clinical and paraclinical data are not exhaustive. Of the 60 French patients in our series who suffered from sicca syndrome, 30% showed stigmata of IgG4-related sialadenitis with or without SS. The results of our study suggest that mild clinical forms of IgG4 salivary involvement with a low prevalence of salivary swelling could exist. This disease is probably underestimated in everyday practice and might constitute a nonexceptional cause of sicca syndrome. Some degree of overlap between SS and sialadenitis linked to IgG4 could also occur to a small extent.Our findings suggest that, in cases of sicca syndrome that do not match SS criteria, IgG4 salivary involvement should be considered and immunostaining with the anti-IgG4 antibody of a LSGB sample may be useful for diagnosis. To assess the efficiency of corticotherapy on dryness symptoms of patients with IgG4 salivary involvement would require a larger study, but this therapeutic option is interesting.


This study determined the amount and severity of EARR  after orthodontic treatment with self-ligating (SL) and conventional (Non-SL) brackets. Differences regarding rate of extraction cases, appointments and treatment time were evaluated.213 patients with a mean age of 12.4 ± 2.2 years were evaluated retrospectively. The treatments were performed with SL brackets  or Non-SL brackets . Measurements of the crown and root length of the incisors were taken using panoramic radiographs. Three-factor analysis of variance (ANOVA) was performed for an appliance effect.There was no difference between patients treated with Non-SL or SL brackets regarding the amount (in percentage) of EARR (Non-SL: 4.5 ± 6.6 vs. SL: 3.0 ± 5.6). Occurrence of severe EARR (sEARR) did also not differ between the two groups (Non-SL 0.5 vs. SL: 0.3). The percentage of patients with need of tooth extraction for treatment (Non SL: 8.1 vs. SL: 6.9) and the number of appointments (Non-SL: 12.4 ± 3.4 vs. SL: 13.9 ± 3.3) did not show any differences. The treatment time was shorter with Non-SL brackets (Non-SL: 18.1 ± 5.3 vs. SL: 20.7 ± 4.9 months).This is the largest study showing that there is no difference in the amount of EARR, number of appointments and extraction rate between conventional and self-ligating brackets. For the first time we could demonstrate that occurrence of sEARR does not differ between the two types of brackets. External apical root resorption (EARR) is defined as either a physiologic or pathologic process with the loss of cementum or dentine resulting in a shortening of the root apex. This process is often associated with orthodontic treatment .Since EARR is a serious iatrogenic problem, there has been intensive research about EARR as an adverse effect during orthodontic treatment. As a result many studies have underlined that EARR often develops during treatment with the fixed multibracket appliance -5. AccorFurthermore several other factors have been implicated in the initiation and progression of EARR’s during orthodontic treatment, such as the duration of treatment -11, the The teeth most affected by EARR are the maxillary and mandibular incisors indicating that mechanical factors might play an important role in the development of EARR ,20,25,26The search for improved efficiency in orthodontic treatment with less adverse effects has generated new types of brackets . Self-liBy now there are a few studies, which observed no significant benefits of SL brackets compared to Non-SL brackets regarding the occurrence of EARR, treatment time or number of appointments. However most of these studies are with a small amount of patients or compare brackets of different companies or with different prescriptions between them ,30-34. TThe objective of this study was to determine the occurrence and severity of EARR on maxillary and mandibular incisors during treatment with SL and Non-SL brackets with the same prescription and from the same company. We furthermore aimed to analyze the amount of extraction cases, number of appointments and treatment time.For this retrospective study 213 patients, being treated in a private praxis between 2008 and 2012, were included by the following criteria:Inclusion criteria:– completed treatment with a multibracket appliance– presence of panoramic radiograph before and after treatment– completed root growth of the maxillary and mandibular incisors before treatment– no evidence of EARR of the maxillary and mandibular incisors on the pretreatment panoramic radiograph– no severely dilacerated incisor roots– caries-free maxillary and mandibular incisorsExclusion criteria:– impacted teeth– trauma before and during active treatment– bends of first, second or third order in the archwire– multiple agenesis– need for orthognathic surgery– endodontic treatment– fixed class II appliance139 patients  were treated with SL brackets . 74 patients  functioned as a control group treated with Non-SL brackets . Both bracket types provided 0.022 slots sizes and MBT prescriptions. Treatments of all patients were performed by the same practitioner with a general arch-wire sequence of a 0.015 twistflex (stainless steel), 0.016 nickel-titanium, 0.016 × 0.022 nickel-titanium, 0.017 × 0.025 nickel-titanium and 0.019 × 0.025 stainless steel . Ligation in the control group was performed with steel ligatures for the conventional brackets.EARR was defined as any reduction in the radiographic lengths of the maxillary and mandibular incisor teeth from the tip of the incisal edge to the apex of the root. Quantitative measurements of the crown and root length of the maxillary and mandibular central and lateral incisors were taken. Any image distortion between the pre- and post-treatment radiographs was calculated using the crown length registrations. This measurement method was established by Linge and Linge and has been described by several studies ,27 Figu.Correction Factor (CF) = C1/C2C1 = Crown length on pretreatment radiographC2 = Crown length on post-treatment radiographThe EARR in millimeter was calculated as following.EARR = R1 – (R2 x CF)R1 = Root length before treatmentR2 = Root length after treatmentIt was decided to express the EARR as relative root resorption (rRR) seen as the percentage shortening per tooth.A tooth is defined as exhibiting a sEARR, when the grade-IV root resorption according to Malmgren et al. is present . In thisPre- and post-treatment radiographs were present due to the usual treatment procedure in Germany, where it is required for diagnostic and treatment procedure. All panoramic radiographs were taken by the same operator. No radiographs were taken out of study reasons and all data were analyzed anonymously to protect the rights of the patients. All patients were informed about using their data for research and gave their informed consent.Statistical analyses were performed using SPSS 20.0  and SAS 9.2 . All multivariable statistical models were adjusted for gender, age, and treatment duration. The two main research questions comprised:(1)  The association of SL brackets with EARR. This was investigated using a repeated measures mixed linear model with fixed and random effects. The model was further adjusted for extraction, treatment mechanism, type of tooth, and interaction between tooth location and self-ligating brackets.(2)  The impact of SL brackets on the occurrence of sEARR. For this we used generalized estimating equations to fit a repeated measures binary logistic regression model.The global significance level of 0.05 was adjusted using a Bonferroni correction which led to local significance levels of α = 0.025.Secondary research questions included the association of SL brackets with (a) the number of visits within a treatment, (b) the duration of treatment, and (c) the number of extraction cases. This was analyzed using multivariable ANCOVA models additionally adjusted for extraction and treatment mechanic and a binary logistic regression model.These analyses can be regarded as explorative and respective p-values as descriptive.To determine systematic error, each of 10 panoramic radiographs taken before and after treatment were reanalyzed five months after the first assessment, a time period long enough for the original measurements to be forgotten in order to determine statistical error according to Dahlberg’s formula. The error in the panoramic radiographs assessment fell within a very good range of (0.14 mm).Two hundred thirteen patients were included into the study, with 139 (65.3%) allocated for treatment with SL brackets and 74 (34.7%) for Non-SL brackets. For the SL group the treatment sample consisted of 56 (40.3%) males and 83 (59.7%) females and for the Non-SL group it consisted of 23 (31.1%) males and 51 (68.9%) females. Overall 1704 teeth were measured before and after treatment  Table .The orthodontic treatment of the group with Non-SL brackets took on average 18.1 ± 5.2 months, the treatment of the group with SL brackets 20.7 ± 4.9 months Table . Thus, tTable The present retrospective study was intended to determine the amount of EARR, as well as the occurrence of sEARR on maxillary and mandibular incisors in a large amount of patients treated with SL or Non-SL brackets.Extraoral x-rays such as panoramic radiographs are considered to be less precise than periapical radiographs for the determination of EARR. The main disadvantage of panoramic radiographs is an overestimation due to the magnification of the radiographs up to 20%. In the anterior region, where EARR was measured, the difference between two radiographs was observed to be less than 0.2 mm . TherefoThe quantity of patients in our study was in comparison to previous studies very large, giving the possibility to display a higher value of significance. Furthermore all patients were treated by one practitioner with the same bracket prescription and the same sequence of archwires, which offered a very good comparability.The amount of EARR in the present study showed no statistically significant difference after orthodontic treatment with the SL or Non-SL brackets. Pandis et al. also did not observe a significant difference between SL and Non-SL brackets in the amount of EARR . AlthougTo evaluate the occurrence of sEARR with a statistical relevance, a large amount of patients is needed due to the rare appearance. Sehr et al. managed to achieve statistical significant results about the occurrence of sEARR  in patients treated with fixed orthodontic appliances. They could show, that 0.5% of orthodontically treated teeth were affected by sEARR . Thus, tIn the present study the central incisors of the mandible were most often affected by sEARR. Most of the previous studies show, that the maxillary and mandibular incisors are more affected by EARR. This might be caused by higher mechanical load due to their smaller root surfaces. The observations of different studies vary equally between central or lateral incisors being more affected by EARR ,19,20,27Treatment time of the two groups differed, whereby the treatment time was 2.6 months shorter with Non-SL brackets compared to SL brackets. This is in concordance with the randomized clinical trial of Fleming et al. . They anNumber of appointments differed slightly between the two groups but without statistical relevance in the multivariable analysis. This finding is similar to the results of Fleming et al., who observed two more appointments for the group of patients treated with SL brackets compared to Non-SL brackets . In thisCompanies promote a better alignment with less need of extraction when fixed appliances with SL brackets are used. We compared the number of extraction cases of both groups of patients and could not detect any difference in the number of extraction cases. Ong et al. analyzed the initial alignment of patients treated with SL and Non-SL brackets and focused on the changes of intercanine and intermolar width. SL brackets did not show a higher efficiency in initial alignment and changes of the arch dimension during treatment were similar between SL and Non-SL brackets . Taken tAs far as we know this is the largest study by now, showing that there is no significant difference in the amount of EARR between patients treated with SL or Non-SL brackets. Furthermore this is the first study showing that there is no difference in the occurrence of sEARR between the two types of brackets.The following conclusions can be drawn from this clinical investigation:1.)  There was no statistically significant difference in the amount of EARR and the occurrence of sEARR between the two types of brackets.2.)  Central mandibular incisors were mostly affected by sEARR.3.)  Number of appointments did not display any difference between Non-SL and SL brackets, whereas treatment time with SL brackets was almost three months longer, which was statistically relevant.4.)  There was no evidence for a difference in the amount of extraction cases in the two groups.The authors declare that they have no conflict of interests.CJ carried out the conception of the study. He supervised the measurements, assembled the data, conducted the interpretation of the data, and drafted the manuscript. PFG did the measurements of the panoramic radiographs. VJ was involved in conception and design of the study, also in analysis and interpretation of data, and drafting the manuscript. MH analyzed the data, did the statistics and helped with the manuscript. DM was involved in conception and design of the study, especially the correct interpretation of the radiographs. HW conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


A 74-year-old man was hospitalized for diabetic nephropathy evaluation and assessment of the effect of treatment on his tubulointerstitial nephritis (TIN). When he was 62 years old, he developed polyarthralgia and had superficial lymph node swelling, mildly increased serum creatinine concentration, hypergammaglobulinemia, hypocomplementemia, high serum IL-2R level, and positive titer of antinuclear antibody. Several tissues were biopsied. Mild chronic sialadenitis and reactive lymphadenitis were identified. Renal specimen showed mild glomerular ischemia, extensive storiform fibrosis, and abundant infiltrating monocytes and plasma cells. He was treated with oral prednisolone and cyclophosphamide. After the treatment, most of his clinical parameters quickly returned to within the reference range. However, he developed diabetes mellitus soon after steroid therapy. At the time of rebiopsy, a high level of serum IgG4 was detected. The second renal biopsy showed diabetic nephropathy without any tubulointerstitial damage. The first biopsied tissues were retrospectively investigated. Large numbers of IgG4-positive plasma cells were detected in the kidneys and lymph nodes. A retrospective diagnosis of IgG4-related TIN with lymph node involvement was made. In conclusion, this paper describes a retrospectively diagnosed case of IgG4-related TIN with lymph node involvement, showing good clinical and pathological prognosis. With the increase in reports of cases of IgG4-related diseases (IgG4-RDs) such as IgG4-related kidney disease (IgG4-RKD), autoimmune pancreatitis, sialadenitis, and retroperitoneal fibrosis –9, the AHere, we report a case that was diagnosed as TIN with interstitial fibrosis 12 years ago and was retrospectively diagnosed as IgG4-related TIN with lymph node involvement. Meaningfully, the storiform fibrosis and infiltrating cells disappeared in the second renal biopsy specimen. A 74-year-old Japanese man was hospitalized in 2008 for renal rebiopsy in order to diagnose diabetic nephropathy and to evaluate the effect of treatment on his TIN. Twelve years ago, when he was 62 years old, he was transferred to our hospital for polyarthralgia, especially on the shoulders and knees, and high gammaglobulinemia. His blood pressure was 170/82 mm Hg. Several lymph nodes ranging from 1 to 2 cm in diameter were palpated on the neck and axillary cavity, without tenderness. The abnormal clinical parameters are presented in D-glucosaminidase (NAG) decreased to 8.1 U/l and no longer exceeded the reference range. One month after the treatment, serum total protein and albumin returned to within the reference range , although some fluctuations were observed during followup. Serum creatinine concentration decreased to 0.9 mg/dL and erythrocyte sedimentation rate (ESR) to 1 mm/h without subsequently increasing. Three months later, serum C1q binding immune complex decreased to <1.5 μg/mL. Five months later, antinuclear antibody (ANA) became negative. Urine protein showed no obvious change during the followup period and remained negative. Hematuria was absent or in trace amount throughout the followup. Serum BMG fluctuated around the upper range value. Urine BMG increased from the 16th follow-up year. The patient was given oral prednisolone treatment starting with a dose of 30 mg/day, tapered 5 weeks later. Cyclophosphamide was started, at 50 mg/day, as a combination treatment after 8 months of prednisolone use. The cyclophosphamide dose was decreased 1 year later, and continued for a total of 2 years. Prednisolone was used for a total of 13.5 years . After t2. The tissue section for light microscopic examination contained 27 glomeruli, 2 of which were sclerosed globally. The other glomeruli showed mild capillary thickening and mild mesangial proliferation. Hyalinosis was observed in some afferents and arterioles (Soon after the use of prednisolone, however, the patient developed diabetes mellitus. A corresponding treatment was given and the prednisolone was changed to cyclophosphamide, but his serum glucose was not well controlled. To estimate the effect of treatment and to survey the existence of diabetic nephropathy, a second renal biopsy was performed. At the time, his blood pressure was 124/66 mm Hg. Physical examination was normal. Laboratory findings showed a serum glucose level of 230 mg/dL and a urine glucose of 2+. The other parameters are shown in terioles . No tubuterioles . Immunof2. A retrospective diagnosis of IgG4-related TIN with lymph node involvement was made. As a retrospective investigation, the first biopsy tissues were subjected to IgG subclass immunostaining with monoclonal antibodies. Salivary gland specimen showed 5-6 IgG4-positive plasma cells/HPF, about 30% of IgG4/IgG-positive cell ratio, and no storiform fibrosis. Lymph node biopsy specimen showed more than 100 IgG4-positive plasma cells/HPF and storiform-like fibrosis . Renal sReports about cases of IgG4-RD have been increasing recently. The All Japan IgG4 team collects all such cases and has defined the concept of this novel entity. By its definition, IgG4-RD is characterized by organ enlargement or nodular/hyperplastic lesions in various organs, concurrently or metachronously, owing to marked infiltration of lymphocytes and IgG4-positive plasma cells, as well as fibrosis of unknown etiology . IgG4-RKMost IgG4-RD patients show multiorgan involvement. The present patient also showed multiorgan symptoms involving the kidneys and lymph nodes. However, the involvement of the salivary gland, which is reported to be an organ susceptible to IgG4-related TIN , 7, coul2, no cell infiltration and fibrosis was observed. Moreover, the pericapsular and perivascular fibrosis identified at the first biopsy was not detected at the second biopsy. All the above results suggest clinical and pathologic improvement in this case. IgG4-RD always shows good response to treatment, mainly steroids , 8. TsubFibrosis is considered a histopathological indicator of poor prognosis –13. EarlIn conclusion, this is a retrospectively diagnosed case of IgG4-related TIN with lymph node involvement that showed good clinical and pathological prognosis by steroid and immunosuppressant treatment.


According to UNAIDS estimates, there are now 33.3 million people living with HIV, including 2.5 million children. During 2009 some 2.6 million people became newly infected with the virus and an estimated 1.8 million people died from AIDS. By mid 2011, 23125 people were infected with HIV, from which, 4311 people died in Iran. The vast majority of people with HIV and AIDS live in lower- and middle-income countries. But HIV today is a threat to men, women and children on all continents around the world. Since there is no vaccine for AIDS and there is no certain cure for AIDS, curing, prevention and secondary infection is essential. This study was conducted to determine the level of knowledge on AIDS and the way of preventing it among faculty members of Zanjan branch, Islamic Azad University.This descriptive study involves 90 people. Data was collected by a questionnaire in five parts . Analysing of data was conducted by SPSS software.Findings revealed that awareness of faculty members of Islamic Azad University of Zanjan about transmission of disease was 72.2, regarding pathology of disease was 67.7, related to complication of disease was 73.3, and about prevention of disease was 74.4.Finally, the level of knowledge of faculty members of Islamic Azad University of Zanjan was higher than moderate (average %70).It can be concluded that the rate of awareness is almost satisfactory but it is not enough and it is necessary to perform educational programs. Key Words: AIDS, Faculty member, Knowledge, Prevention.


Small cell lung carcinoma (SCLC) has poor prognosis and remains orphan from targeted therapy. MET is activated in several tumour types and may be a promising therapeutic target.MET gene status and protein expression were evaluated in a panel of SCLC cell lines. The MET inhibitor PHA-665752 was used to study effects of pathway inhibition in basal and hepatocyte growth factor (HGF)-stimulated conditions. Immunohistochemistry for MET and p-MET was performed in human SCLC samples and association with outcome was assessed.To evaluate the role of MET in SCLC, n=77), respectively. MET phosphorylation was associated with poor median overall survival (132 days) vs p-MET negative cases (287 days)(P<0.001). Phospho-MET retained its prognostic value in a multivariate analysis.In MET mutant SCLC cells, HGF induced MET phosphorylation, increased proliferation, invasiveness and clonogenic growth. PHA-665752 blocked MET phosphorylation and counteracted HGF-induced effects. In clinical samples, total MET and p-MET overexpression were detected in 54% and 43% SCLC tumours (MET activation resulted in a more aggressive phenotype in MET mutant SCLC cells and its inhibition by PHA-665752 reversed this phenotype. In patients with SCLC, MET activation was associated with worse prognosis, suggesting a role in the adverse clinical behaviour in this disease. Small cell lung carcinomas (SCLCs) account for ∼15% of lung cancers  disulphide bonded to a membrane-spanning β- (145 kDa) chain .A panel of 10 SCLC cell lines models was screened for MET alterations for this study. The SCLC cell lines H69, H69AR, H187, SHP-77, H345, H740, H748, H865, and H524 and H1688, and the NSCLC H1993 cells were obtained from the American Type Culture Collection (ATCC) . All cells were cultured according to the ATCC instructions (μg ml−1 at −80°C. Treatments (unless otherwise indicated) were done at 40 ng ml−1 as final concentration in culture medium.Recombinant human HGF was purchased from Calbiochem  and was resuspended in sterile PBS containing 0.1% BSA and stored at a stock concentration of 100 M in DMSO at −80°C. The stock solution was diluted for instant use in our experiments.PHA-665752, (3Z)-5-[sulfonyl]-3-[methylene]-1,3-dihydro-2H-indol-2-one  was dissolved to a stock concentration of 10 mThe following antibodies were purchased from the manufacturers listed below and used for western blot assay: Met (25H2) mouse monoclonal antibody (mAb), p-MET Y1234/1235 (3D7) rabbit mAb, p-MET Y1349 (130H2) rabbit mAb, p-MET Y1234/35 (D26) XP rabbit mAb, p-GAB1 Y307-rabbit polyclonal antibody (pAb), ERK1/2 rabbit pAb, p-ERK1/2 (Thr202/Tyr204) pAb were obtained from Cell Signaling  and GAB1 (H198) rabbit pAb from Santa Cruz Biotechnology . Western blot from whole-cell extracts was performed as previously reported (MET gene in cell lines was assessed by fluorescence in situ hybridisation (FISH) using the ON·C-MET (7q31)/SE 7 FISH probes , labelling the centromeric alpha-satellite region, specific for chromosome 7 (spectrum green), and the 7q31 region that contains the MET gene (spectrum orange), as described  was performed by direct sequencing. Primers for PCR amplification and sequencing were designed using the Primer Express software  and were as follows: 5′-GCAGCAGCAAAGCCAATTTAT-3′ and 5′-TGACTTTGGCTCCCAGGGC-3′ for the E168 and 5′-ACCCATGAGTTCTGGGCACT-3′ and 5′-CAGAACAATAAACTGAAATATACCTTCTGG-3′ for the R988 and T1010. PCR conditions were as follows: 95°C 10 min 1 cycle; 95°C 1 min, 55°C for 1 min, 72°C for 1 min, 40 cycles; and 72°C 10 min, 1 cycle. Sequencing was performed with BigDye v3.1 (Applied Biosystems) following the manufacturer's instructions and analysed on a 3500Dx Genetic Analyzer (Applied Biosystems).For mutational studies of tumour samples, DNA was extracted from macrodissected tumoural paraffin-embedded tissue using the QIAamp Tissue Kit  according to the manufacturer's protocol. The mutational analysis of the 5 cells per well in a six-well plate with culture medium containing 10% FBS. After 24 h HGF, PHA-665752 or the combination were added at 40 ng ml−1 or 0.5 μM, respectively, and incubated during 72 h. Cell viability was determined by trypan blue/haemocytometer exclusion method. Each experimental condition was done in duplicate. The results were plotted as percentage of control.To measure effects of HGF, PHA-665752 or the combination on the viability of SCLC cell lines, we seeded 3 × 104 cells in 35 mm plates) were grown in 0.3% agar containing FBS 10% in RPMI 1640 medium in the presence and absence of HGF (40 ng ml−1) and PHA-665752 (0.5 μM) on top of 0.5% agar. The plates were incubated  and cells were fed every 3 days with 0.5 ml of culture medium containing the above-mentioned HGF and PHA-665752 concentrations. The number and size of colonies were evaluated at × 20 magnification on a light microscope at each condition after 21 days.Single cell suspensions (2 × 104 adherent cells or 1 × 106 cells growing in suspension were seeded into the inserts of 24-well (8 μm pore size) CHEMICON Invasion Chamber  and treated with PHA-665752 (0.5 μM), a concentration that does not have a significant effect on cell viability. Inserts were placed into Falcon companion plates  containing 10% FBS and 40 ng ml−1 HGF and incubated for 24 h at 37°C and 5% CO2 atmosphere.To evaluate the invasiveness of SCLC cell lines we prepared cell suspensions in serum-free media and 2.5 × 10μl per well) was applied to dissolve the stained cells, and then the dye/solute mixture was transferred to a 96-well plate for colorimetric reading of optical density (OD) at 560 nM. The OD value represents the invasive ability. All experiments were performed in triplicate.Following incubation non-invading cells were removed from the top chamber using cotton swabs and the outside of the insert was gently rinsed with PBS, stained with 0.25% crystal violet for 20 min and rinsed again. Acetic acid  (Santa Cruz Biotechnology). We performed two series of experiments to rule out unspecific staining both in cell lines and tumour samples. First, in both formalin-fixed basal and HGF-treated H69 cell pellets  mouse mAb , MET (SP44) mouse mAb , p-MET Y1349 (130H2) rabbit mAb, p-MET Y1234/35 (3D7) rabbit mAb, p-MET Y1234/35 (D26) XP rabbit mAb  , slides were incubated with primary antibody for 1 h at a dilution of 1 : 50 for 3D4 MET, 1 : 1 MET SP44 mouse mAb, 1 : 20 for p-MET Y1349 and 1 : 50 for p-MET Y1234/35. Then, sections were incubated with the specific polymer EnVision Flex+ (Dako), revealed with 3–3′ diaminobenzidine, a chromogen, and counterstained with haematoxylin.The study population consisted of 77 patients diagnosed with SCLC at any stage from whom we had clinical and follow-up information. Biopsies were obtained before treatment and subsequently patients initiated standard chemotherapy regimes. All samples except for two were non-surgical core biopsies as surgery is not a standard procedure in SCLC treatment . Specimens were retrospectively retrieved from Parc de Salut Mar Biobank  and Fundación Jiménez Díaz Biobank . This study was approved by the institutional review board of each participating centre. Three-Sections were evaluated by two pathologists independently blinded to clinical information on a light microscope . The MET and p-MET staining was scored when any percentage of tumour cells was stained in the membrane. A semiquantitative histoscore (Hscore) was calculated, determined by estimation of the percentage of tumour cells positively stained with low, medium, or high staining intensity for each marker. The final score was determined after applying a weighting factor to each estimate. The formula used was Hscore=(low %) × 1+(medium %) × 2+(high %) × 3, and the results ranged from 0 to 300. Staining of non-tumour epithelial cells  was also assessed.χ2-test (Fisher's exact test). Correlations between expression scores by different primary antibodies were calculated by Spearman rho test. Overall survival was analysed by the Kaplan–Meier method. Curves were compared by the log-rank test. Multivariate analysis was performed using the Cox proportional hazards model, including the variables that had reached statistical significance in the univariate analysis. All the statistical tests were conducted at the two-sided 0.05 level of significance. Receiver operating curve (ROC) was used to determine the optimal cutoff point for MET and p-MET overexpression . To analyse correlations between MET status and clinical-pathological variables we used the pression . As showMET amplification in the NSCLC H1993 cell line as previously reported (data not shown). We therefore used this cell line as a positive control of MET activation. In both H69 and H69AR (chemoresistant) we confirmed the reported juxtamembrane mutation R988C on exon 14 of the MET gene (data not shown)  showed lack of MET expression. Based on these results we selected H69 as a MET mutant model and H524 (no expression of MET), H187 (MET expression) and H345 (MET expression) as models of wild-type MET cell lines. Results with H69 were confirmed with H69AR .To characterize MET status in our cell lines, we performed FISH to study the gene copy number, sequencing to study the mutations described on exon 14 for SCLC and protein expression by western blot. None of the 10 SCLC cell lines showed MET amplification. We found MET amplified H1993 cell line, showed high levels of basal and phosphorylated MET and absence of modulation by HGF 40 ng ml−1. μM (data not shown).To further characterize the MET pathway in these selected cells, we performed a western blot analysis of total and phosphorylated MET, and downstream relevant molecules, ERK and GAB-1. As expected, our positive control, the MET amplified cell line. There was increased proliferation upon HGF treatment in both mutant H69 and H69AR cell lines (30–60% increase) and this effect was prevented by PHA-665752 at 0.5 μM. Neither the proliferative effect nor the inhibitory effects were observed in the MET wild-type H524, H345 and H187 cell lines (We then assayed the effects of HGF and PHA-665752 or the combination on cell viability in selected cell lines. PHA-665752 treatment inhibited growth in the H1993 ll lines .−1) doubled colony formation in soft agar. Consistent with the observation that H69 is dependent on MET signalling, PHA-665752 at 0.5 μM reduced colony formation in H69 cells (P<0.05). This effect was also observed in H69AR and correlated with inhibition of MET phosphorylation (data not shown). In contrast, these effects were not observed in the H187, H345 and H524 wild-type cell line . In contrast, for the MET wild-type H187, H345 and H524 invasion was not influenced by treatment with PHA-665752 . Four were from regional lymph nodes and one from a distant site. Patients’ characteristics are listed in As MET phosphorylation seems to be a good indicator of the activation of the MET pathway, we explored the implication of the expression of this marker in tumour samples. We sought to evaluate the pattern and prevalence of total MET and p-MET expression in a series of 77 human SCLC samples. The vast majority of the assayed specimens were from primary tumours . The MET phosphorylation was assessed using two antibodies: Y1349 (docking site of MET) and Y1234/35 (autophosphorylation site of MET). Results in P<0.001, R2=0.73) between both antibodies. Strong agreement was detected in MET and p-MET scores between two observers  and mean of values were considered for statistical analysis. The ROC was used to determine the optimal cutoff points for MET and p-MET overexpression, which were calculated at Hscore of 120 and Hscore of 5, respectively. At this value, the sensitivity of the test for MET was 57.1%, with a specificity of 62.7%, and the sensitivity for p-MET was 46.3%, with a specificity of 85.7% .The MET expression in SCLC samples was consistent using two different primary antibodies (3D4 and SP44)  specimens showed any grade of MET expression and 42 (54.5%) were considered as tumours with MET overexpression. The MET staining was consistently observed in the membrane of tumour cells, and the expression was diffuse in the tumour. Differences in intensity of MET expression were occasionally detected in the same tumour.Thirty-three (42.9%) cases showed p-MET overexpression following the ROC criteria. All p-MET positive cases expressed the total form of the receptor. The pattern of p-MET staining was membranous and varied from a few cells showing positivity to diffuse staining in other tumours .Sixty-eight cases had also histologically normal bronchial epithelium representation in the section. Total MET staining was faintly detected in non-tumour epithelial cells in 67 (98%) specimens and p-MET staining in 16 (24%) .P=0.048) we found was between p-MET positivity and extensive stage . Performance status and gender were not significantly linked to survival in our series in the univariate analysis.To provide data regarding the prognostic impact of MET expression and activation in human SCLC, we performed survival analysis of our series of patients stratified by the status of the markers. We first evaluated the association of clinical variables with overall survival (OS). As expected, patients with limited stage had a significantly better survival than patients with advanced disease (P=0.163)  (P=0.163) .P=0.001). Those patients with p-MET overexpression had worse prognosis (OS: 132 days) compared with p-MET negative/low expression cases (OS: 287 days). Moreover, an inverse significant correlation was detected between time of survival and levels of p-MET expression (P=0.022). The range of results for Hscore for p-MET was from 0 to 150. There was a trend towards an association between higher score and worse outcome (P=0.091). Phospho-MET was assayed with Y1349 in the whole series (vs 270 days)  . This suP<0.05).Finally, in a multivariate analysis including disease stage   and p-MEMET mutations and p-MET staining were related, we analysed the previously described MET mutations in SCLC samples and cell lines: R988C, T1010I and E168C in our specimens. The 37, 20 and 33 samples with enough remaining material were evaluated for each mutation respectively. No mutations were found in these codons, thus precluding the analysis of their potential relationship with p-MET. Of note, however, MET mutations have been described in human SCLC and larger series may allow to analyse this correlation.To explore whether MET mutant SCLC cells but not in MET wild-type SCLC cells. The magnitude of the effects was greater in terms of reduced clonogenicity and invasiveness when the inhibitor was used at relatively low concentrations, consistent with those needed to inhibit the target. Furthermore, we provide novel evidence for the activation of MET in about 40% of patients with SCLC and a link with poor outcome, independent of disease stage.Here we demonstrate that MET inhibition, using PHA-665752, is able to counteract HGF-induced effects in MET mutation, basal levels of phosphorylation were low in both cell lines and only when stimulated with HGF this phosphorylation increased significantly as well as the oncogenic phenotype. Previous studies have suggested that stimulation with HGF is necessary to display the oncogenic properties of cells carrying MET mutations, which is in line with our findings and could be considered an example of oncogene expedience  and MET pathway activation via exogenously added HGF have an increased proliferation, invasiveness and colony formation capacity. Some of these effects have been previously described for SCLC cell lines  MET expression was not associated with prognosis in our series. In fact, in the literature, the prognostic role of total MET in human cancer is controversial. Albeit many studies point to an adverse prognostic role (Heterogeneity in MET expression in whole tissue sections in other tumours has been described (MET mutations in SCLC in a subset of the whole population. No mutations were found in this sample set. However, MET mutations have been previously described in SCLC specimens (Finally, in order to correlate our preclinical work with our clinical findings we performed mutational studies in cases with available tissue. Due to limited material we were able to study only previously described MET-activating mutations. They also argue in favour of testing MET targeting agents preferentially in patients whose tumours display p-MET expression or MET mutations.In conclusion, our results add support to the view of MET as an attractive therapeutic target for a subgroup of SCLCs-harbouring


The PLZF/RARA fusion protein generated by the t translocation in acute promyelocytic leukaemia (APL) is believed to act as an oncogenic transcriptional regulator recruiting epigenetic factors to genes important for its transforming potential. However, molecular mechanisms associated with PLZF/RARA-dependent leukaemogenesis still remain unclear.We searched for specific PLZF/RARA target genes by ChIP-on-chip in the haematopoietic cell line U937 conditionally expressing PLZF/RARA. By comparing bound regions found in U937 cells expressing endogenous PLZF with PLZF/RARA-induced U937 cells, we isolated specific PLZF/RARA target gene promoters. We next analysed gene expression profiles of our identified target genes in PLZF/RARA APL patients and analysed DNA sequences and epigenetic modification at PLZF/RARA binding sites. We identify 413 specific PLZF/RARA target genes including a number encoding transcription factors involved in the regulation of haematopoiesis. Among these genes, 22 were significantly down regulated in primary PLZF/RARA APL cells. In addition, repressed PLZF/RARA target genes were associated with increased levels of H3K27me3 and decreased levels of H3K9K14ac. Finally, sequence analysis of PLZF/RARA bound sequences reveals the presence of both consensus and degenerated RAREs as well as enrichment for tissue-specific transcription factor motifs, highlighting the complexity of targeting fusion protein to chromatin. Our study suggests that PLZF/RARA directly targets genes important for haematopoietic development and supports the notion that PLZF/RARA acts mainly as an epigenetic regulator of its direct target genes. RARA) gene on chromosome 17, finds itself involved in chromosomal recombination with one of several different potential partner genes (designated “X”), i.e. PML, PLZF, NuMA, NPM1, FIP1L1, PRKAR1A, STAT5b or BCOR, generating X/RARA fusion proteins Retinoic acid receptors (RARs) belong to a family of nuclear receptors that can activate or repress transcription of target genes by recruiting co-activator or co-repressor complexes. It is the binding of its natural ligand, retinoic acid (RA), which transforms the activity of RAR from a repressor to an activator of transcription by inducing a conformational change in the ligand binding domain structure all-trans RA (ATRA) and arsenic trioxide (ATO) de novo target genes due to the sequence-specific DNA binding activity of PLZF. In addition, the situation is made more complicated by expression of the reciprocal RARA/PLZF that can potentially bind to PLZF binding sites where it may function as a transcriptional activator The nature of the fusion partner X has a decisive impact on the disease state and sensitivity to the therapeutic effects of Here, we report a genome-wide ChIP-on-chip study of PLZF/RARA gene targets using an inducible cell system. We identify 413 high-confidence specific target genes that provide clues as to how PLZF/RARA contributes to APL pathogenesis.HOXD cluster HOXD4 and HOXD9 genes in both U937-PLZF/RARA and control cell lines. These data were corroborated by quantitative PCR analyses of independent PLZF ChIP samples (ChIP-qPCR) (RARB2 (the RARA fusion model target gene) as well as C/EBPE, ASB2, PRAM1 and IL8, that have all previously been reported to be regulated by X/RARA fusion proteins To identify gene targets directly regulated by PLZF/RARA, we took advantage of a zinc-inducible U937 cell system either expressing the PLZF/RARA fusion protein or harbouring an empty vector IP-qPCR) . To idenIP-qPCR) . Among tbona-fide binding regions. Functional classification of specific PLZF/RARA target genes according to gene ontology (GO) analyses showed that PLZF/RARA binds to genes implicated in either basic cellular processes or developmental related processes . A highly significant 20% of the genes targeted by PLZF/RARA have altered expression patterns  with 11% becoming down regulated and 9% being up regulated upon PLZF/RARA binding  IDH2, PAX5 and DAZAP1. IDH2 is frequently mutated in acute myeloid leukaemia and its mutations confer adverse prognosis in cytogenetically normal AML PAX5 and DAZAP1 are both involved in translocations associated with acute lymphoblastic leukaemia To determine the transcriptional effect of PLZF/RARA on its identified target genes, gene expression profiling was performed in U937-MT (control) and U937-B412 (PLZF/RARA) cell lines. Expression profiling following PLZF/RARA induction was evaluated by microarray analysis and 4321 genes were identified with altered expression levels  and RARB2 repression  PLZF/RARA has conserved the DNA binding domain of RARA that binds to RA response elements (RAREs) defined by a direct repeat of the core motif By comparing PLZF bound regions between U937 and PLZF/RARA-induced U937 cells we identified 413 specific PLZF/RARA target genes. This selection of bona-fide PLZF/RARA targets was significantly enriched in genes important for hematopoietic development and included genes effectively downregulated in primary PLZF/RARA+ APL cells. Moreover, PLZF/RARA binding was preferentially associated with gene repression at sites enriched for the repressive H3K27me3 histone modification mark. Analysis of PLZF/RARA bound sequences suggests PLZF/RARA binds canonical and degenerate RARE motifs, and recruitment of PLZF/RARA may also occur via other hematopoietic specific TFs. This study helps to clarify the role of the PLZF/RARA fusion protein as an oncogenic transcriptional regulator during leukaemogenesis and provides further insights into the pathogenesis of the disease.4 for at least 24 h.Human myelomonoblastic cell lines U937-MT and U937-B412 Total RNA was extracted using the RNeasy Mini kit according to the manufacturer's protocol (Qiagen), For cDNA synthesis, reverse transcription was performed with 2 µg of the total RNA, oligo dT, dNTPs, DTT, buffer and Superscript Retrotranscriptase II (Invitrogen). cDNA was analyzed by qPCR using Brilliant SYBR Green Master Mix (Stratagene) according to the manufacturer's instructions with the 7500 Fast Real Time PCR system (Applied Biosystems). Primers used for real-time PCR are given in Supporting Information.7 cells per condition was used. For ChIP using H3K27me3 , H3K4me3  or H3K9K14Ac , chromatin from 5×106 cells per condition was used. Chromatin was diluted 5× in 150 mM NaCl, 10 mM Tris-HCl pH 8.0, 1% TritonX-100, 0.1% sodium deoxycholate (NaDOC), 1 mM EDTA, 0.5 mM EGTA. Chromatin, precleared 1 h with protein G-coupled magnetic beads, was incubated overnight at 4°C using the different antibodies followed by an incubation for 2 h with blocked protein G-coupled magnetic beads . Real-time PCR was performed using Brilliant SYBR Green Master Mix (Stratagene) according to the manufacturer's instructions with the 7500 Fast Real Time PCR system (Applied Biosystems). Primers used are given in Chromatin Immunoprecipitation (ChIP) was essentially performed as described before by Boukarabila et al. Total DNA and ChIP DNA were amplified using the whole genome amplification kit  and fluorescently labelled using BioPrime Array CGH Genomic labelling kit (Invitrogen). Labeled samples were hybridized following the manufacturer's instructions to a custom promoter array  containing 236.992 probes spanning the promoter regions  of 18.000 refseq genes (UCSC assembly HG17). Experiments were performed in duplicate and showed high correlation (R≥0.769). All data is MIAME compliant. Median-normalized log2 enrichment ratios (ChIP/Input) of merged replicates were calculated using CoCAS software Target genes were classified using DAVID http://www.perl.org) on ChIP-on-chip peak sequences. The expression code was built according to the known repeat pattern (A/G)G(G/T)TCA. We allowed a gap ranging from 0 to 100 bases and performed the calculation for finding DR, IR and ER on both strands. Analyses of transcription factor binding site enrichments were performed using MatInspector tool from GENOMATIX software (http://www.genomatix.de).We looked for the consensus RARE motif using Perl regular expressions (P<0.05). For patient samples, microarray raw data was taken from Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/projects/geo/), under the data set GSE8510 for primary PLZF/RARA+ APL patient cases and under the data set GSE15061 for normal bone marrow samples (NBM). Details of the patient samples analysed have been reported previously MT and B412 cells were cultured in duplicate in the presence of zinc for 48 hours. RNA was extracted using QIAGEN RNeasy and quality was tested using an Agilent Bioanalyzer. 1 µg of RNA was hybridized to U133 Plus 2.0 arrays (Affymetrix). Affymetrix U133A.CEL data files from B412 cells and MT cells were imported into R and probe intensities were normalized using GC-RMA. A total of 4321 genes were found to be differentially expressed  and U937-B412 (2) treated with zinc and were immunobloted for PLZF/RARA and PLZF (α-PLZF) or RARA (α-RARA).(TIF)Click here for additional data file.Figure S2Transcription factor binding sites over-represented in PLZF/RARA target promoters. MatInspector tool from GENOMATIX was used to identified transcription binding sites in PLZF/RARA bound promoters.(TIF)Click here for additional data file.Table S1List of PLZF/RARA target genes. Indicated in the table are symbols and full names of PLZF/RARA target genes, the presence, number and type of RAREh in PLZF/RARA enriched peak and micro array data of PLZF/RARA target genes. Diff mean corresponds to the difference of the mean between MT and B412 cell lines.(XLSB)Click here for additional data file.Text S1Supplemental experimental procedures.(DOCX)Click here for additional data file.


The wild-type Zbtb16 gene encodes a transcription factor that belongs to the POK (POZ and Krüppel) family of transcriptional repressors. In addition to nine Krüppel-type sequence-specific zinc fingers, which make it a member of the Krüppel-like zinc finger protein family, the PLZF protein contains an N-terminal BTB/POZ domain and RD2 domain. PLZF has been shown to be involved in major developmental and biological processes, such as spermatogenesis, hind limb formation, hematopoiesis, and immune regulation. PLZF is localized mainly in the nucleus where it exerts its transcriptional repression function, and many post-translational modifications affect this ability and also have an impact on its cytoplasmic/nuclear dissociation. PLZF achieves its transcriptional regulation by binding to many secondary molecules to form large multi-protein complexes that bind to the regulatory elements in the promoter region of the target genes. These complexes are also capable of physically interacting with its target proteins. Recently, PLZF has become implicated in carcinogenesis as a tumor suppressor gene, since it regulates the cell cycle and apoptosis in many cell types. This review will examine the major advances in our knowledge of PLZF biological activities that augment its value as a therapeutic target, particularly in cancer and immunological diseases.The promyelocytic leukemia zinc finger (PLZF) protein, also known as Zbtb16 or Zfp145, was first identified in a patient with acute promyelocytic leukemia, where a reciprocal chromosomal translocation  The family of zinc finger proteins is composed of regulatory proteins that participate in many molecular and cellular pathways and is considered to be one of the most profuse regulatory protein families in eukaryotic cells, with more than 200 members. Different members of the zinc finger protein family contain multiple cysteine (Cys) and/or histidine (His) residues that require one or more zinc ions to stabilize their structures. The majority of the zinc finger proteins play important roles in DNA binding, RNA binding, RNA packaging, and protein–protein interactions. The family is divided into three sub-groups according to structure and the number of zinc fingers  gene was first described in humans in 1993, where its encoded protein was found to be fused in-frame with the retinoic acid receptor alpha (RARα) in a patient diagnosed with a rare form of acute promyelocytic leukemia (APL) with a reciprocal chromosomal translocation t. The protein was named the promyelocytic leukemia zinc finger (PLZF), because it was the first zinc finger family member exhibiting a pronounced effect on the pathology of promyelocytic leukemia , all encoding functional proteins, and composed of seven exons with shared homology between exons 3 and 6. The first transcript (ENST00000335953) is 2,477 bp in length and encodes a 673 aa protein. The second transcript (ENST00000392996) is 2,249 bp in length and encodes a 673 aa protein. The third transcript (ENST00000310883) is 1,728 bp in length and encodes a 550 aa protein. In mouse, the gene is located in chromosome 9, and is composed of seven exons. However, unlike its human analog, it only has one transcript (ENSMUST00000093852) that is 5,100 bp in length and encodes a 673 aa protein, which is similar in length to the human analog transcripts 2 and 3.2H2 zinc fingers that facilitate sequence-specific DNA binding to its target genes allowing PLZF to act as a transcriptional repressor  gene knockout was developed in 2000 by researchers at Memorial Sloan-Kettering Cancer Center (Cornell University). They replaced the second exon of full-length PLZF with the neomycin resistance gene. PLZF−/− mice displayed major musculoskeletal-limb defects with homeotic transformations of vertebral segments, deformed cartilage and skeleton patterning, and alterations in digit formation that is caused by an inhibition of apoptosis in hindlimb early progenitor cells in the autopod region possibly due to Hox gene regulation , possibly through the PLZF-mediated interaction with polycomb family members such as Bmi-1, to the promoters of the Hox gene clusters affecting only the development of the posterior regions of the limb bud . CBFA1 affects many important factors during the pluripotent differentiation of human mesenchymal stem cells (hMSCs) into the osteogenic lineage such as collagen 1A1 (COL1A1), alkaline phosphatase (ALP), and osteocalcin (OCN). Loss of PLZF expression during the early stages of pluripotent differentiation of hMSCs leads to the disruption of spinal ossification  was lethal as measured by survival curves. However, pretreating the mice with interferon (IFN) for 6 h prior to infection protected the wild-type neonatal mice from infection, with survival times exceeding 3 weeks. On the other hand, mice lacking PLZF expression died after 6 days of infection, with viral loads in their vital organs about a thousand times higher than wild-type mice after only 48 h post-infection. This occurred despite the fact that both genotypes expressed comparable levels of IFNs after infection, showing that activation of certain anti-viral genes was impaired in the absence of PLZF. Similarly, introducing the encephalomyocarditis virus (EMCV) via intraperitoneal injections to wild-type and PLZF−/− mice yielded equivalent results when comparing survival curves between the two genotypes , accounting for the fatal phenotype in PLZF−/− mice. Moreover, IFN-induced activation of natural killer (NK) cells was also impaired in PLZF−/− mice, which was attributed to the marked decrease in CXCL10 expression in NK-cell-rich organs in these animals  or a kinase in the same pathway to be the responsible enzyme. The study demonstrated that PLZF transcriptional activity is enhanced and caused the binding of PLZF to the promoter of many ISGs. Unlike its previously reported transcriptional repression activity, following IFN stimulation, PLZF acted as a transcriptional activator, which was a novel finding for this zinc finger protein family member  proteins are a family of small proteins that reversibly attach to their target proteins to regulate their functions. SUMO-1, a SUMO protein family member, binds to lysine 242 residue in the RD2 domain of PLZF NEUROG1 expression , ubiquitin modulates many biological processes by targeting different proteins in various pathways directing them for biological recycling. The BTB/POZ domain of PLZF was shown to bind BTBD6, a known ubiquitin ligase adaptor protein for the CUL3 ubiquitin E3 ligase complex, leading to its export from the nucleus and subsequently its degradation. The removal of PLZF from the cell was shown to be essential for differentiation of neural progenitor cells during the early stages of neurogenesis, possibly by restoring PLZF gene in 1993, the protein product was subsequently noted to be localized inside the nucleus within specialized nuclear compartments called nuclear speckles  treatment of human fibrosarcoma HT1080 cells caused the metalloprotease cleavage of membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) into its soluble form HB-EGF-C. This soluble form is known to bind the zinc finger motif of PLZF and mediates its nuclear export into the cytoplasm. The decrease in PLZF nuclear level in TPA-treated HT1080 cells led to increased cyclin-A2 levels, a known PLZF-target gene, which was responsible for cell cycle progression into the S-phase , is known to be crucial for the maintenance and differentiation of hematopoietic stem cells and hematopoietic progenitor cells  gene and an up-regulation of tumor protein p53-inducible nuclear protein 1 (TP53INP1), inhibitor of DNA binding 1 (ID1), and inhibitor of DNA binding 3 (ID3); all of which are apoptosis-inducing genes -dependent murine hematopoietic precursor cells to accumulate in the Gin vitro. They also demonstrated how human endometrial and myometrial stromal cells expressed PLZF only during the mid-late secretory phase of the menstrual cycle in vivo, where the levels of GC and progesterone are relatively high , such as cortisone and dexamethasone, are a type of steroid hormones that are synthesized and secreted by the adrenal cortex. They function through the GC receptors, which are found in the vast majority of mammalian cells, to regulate many essential biological functions, such as lipolysis, glycolysis, and cardiovascular and immunological processes. In a DNA microarray analysis using the T47D/A1-2 breast cancer cell line, PLZF was found to be differentially induced by dexamethasone. This induction was suggested to be, in part, the cause of breast cancer cell death after dexamethasone treatment  and HDAC inhibitors (HDACi). The ability to regulate the function of these enzymes to manipulate the DNA transcriptional signal in many disease backgrounds is crucial , they showed that PLZF up-regulated many immune response genes after IFN treatment and that those genes contained putative PLZF-binding sites in their promoter regions. Many ISGs failed to respond to high levels of IFN in PLZF−/− mice after viral infection. The expression levels of Oas1g, CXCL10, Rsad2 (Viperin), and Ifit2 were significantly lower, showing the requirement of PLZF in the induction of the anti-viral response  factors to induce the Janus kinase (Jak)–STAT signaling cascade. As discussed earlier, Xu et al. demonstrated that PLZF was required for the anti-viral innate immune response PLZF−/− mice, although constituting the same percentage of total lymphocytes as compared to the wild-types, were functionally impaired. This was reflected by their failure to spontaneously lyse Yac-1 cells (an NK cell-specific target because of their missing MHC-1). This was also true after poly I:C-mediated IFN activation. Moreover, Granzyme B (GzmB) induction after IFN activation was also affected, with a lower percentage of PLZF−/−splenic NK cells showing GzmB positive staining. This impairment of IFN activation was confirmed to be the direct result of lost PLZF expression, which was required by a specific subset of ISGs. In spite of the well-known role of PLZF as a transcriptional repressor, a novel activator role was described for the first time with regards to the IFN signaling pathway, where phosphorylated PLZF binds to the promoter regions of those ISGs and actively drives their transcription  class-I. PLZF expression affects the effector function of NK cells. NK cells from PLZF−/− mice did not affect the number of mature iNKT cells, but did have a dramatic impact on their function. Most iNKT cells expressed low levels of CD44, CD8, NK1.1, DX5, NKG2D, CD244, CD24, and CD122. Conversely, they expressed high levels of CD69 and failed to remain in the thymus after maturation; instead accumulating in the liver, lymph nodes, and spleen. Functionally, PLZF-deficient iNKT cells failed to secrete both IL-4 and IFNγ after immunological challenge, a hallmark of NKT cells  cells are a heterogeneous population of T cells that both express surface antigen markers designated for NK cells and recognize the Cd1d antigen that presents many glycoprotein and lipid molecules. Invariant NKT (iNKT) cells are a subgroup of NKT cells with distinctive properties. They express an invariant α chain of the T cell receptor (TCR), a specific TCR-β chain that uses the variable region 8.2, and they can respond to immunological stimuli by cytokine secretion within minutes  umbilical human cord blood-derived myeloid progenitor cells that had been transduced with PLZF expression/knockdown vectors. They successfully demonstrated that PLZF was able to regulate the differentiation capacity of these myeloid progenitors. PLZF achieved this through the transcriptional modulation of many key transcription factors involved in myeloid differentiation, such as GFI-1, C/EBPα, LEF-1, DUSP6, and ID2. This demonstrates how PLZF-controlled cellular proliferation in early hematopoiesis is essential in establishing a balance between the numbers of progenitor and fully differentiated cells  between RARα and PLZF. The fusion proteins caused the progression of the leukemia by suppressing many retinoic acid-regulated genes, thereby locking the differentiation of granulocytes into the “promyelocyte” stage. Specifically, the patient had a chromosomal rearrangement that led to a reciprocal translocation where two fusion gene products were produced: PLZF-RARα and RARα-PLZF. PLZF is located in chromosome 11q23 and RARα is located in chromosome 17q21. The first form of the fusion protein “PLZF-RARα” is composed of full-length PLZF until the second zinc finger of the protein, where a breakpoint joins RARα domains B–F. The second fusion protein RARα-PLZF binds the A domain of RARα with the remaining seven zinc fingers (3–9) of PLZF  protein was discovered in a patient with APL . The cause of the disease was found to be a translocation In vitro, dihydroxyvitamin D3 (D3) and transforming growth factor β1 (TGFβ-1) drive the differentiation of the human monocytic U937 cell line. Similarly, dimethyl sulfoxide (DMSO) can induce the differentiation of the human promyelocytic leukemia HL-60 cell line. PLZF-RARα expression blocked the D3-TGF-induced monocytic and DMSO-induced granulocytic differentiation of both U937 and HL-60 cells, respectively  expression , transforming the chromatin in those regions insensitive to remodeling by HDACs. This could explain the non-responsiveness phenotype to all-trans retinoic acid treatment in PLZF-RARα-mediated APL patients  protein was found to interact with the BTB domain of PLZF, reflecting the high expression of PLZF in normal cells and the absence of expression in cancer cells. CCS3 expression followed a similar pattern in human cervical cells. Restoring CCS3 expression in SiHa cervical cancer cells caused a dramatic change in morphology, a reduction in the numbers of viable cells in culture, and increased apoptosis. CCS3 was shown to achieve this by interacting with PLZF to repress PLZF-target genes, such as PLZF expression and that expression could be restored by the ectopic expression of the androgen receptor (AR). Overexpressing PLZF in these cells caused a marked down-regulation of the pre-B-cell leukemia transcription factor (PBX1) and led to decreased proliferation and survival in vitro. However, this was not true in LNCaP cells, which are androgen-dependent prostate cancer cells. This reflects the fact that PLZF-mediated repression is androgen-dependent and indicates how DU145 could escape androgen-based chemotherapy . In vivo interaction between CUX1 and thePLZF promoter was confirmed by electrophoretic mobility shift assay (EMSA) and chip analysis. CUX1 was also able to bind to the promoter region of PLZF in Caco-2/15 and HEK293 cells in vitro. A reporter assay showed that CUX1 blocked the transcriptional repression of PLZF in a dose-dependent manner. When assessing the PLZF gene expression profile in different colon cancer cells, an inverse relation was discovered between PLZF and CUX1 gene expression (Fréchette et al., An analysis of the upstream translated region of human Promyelocytic leukemia zinc finger is a transcription factor implicated in major developmental and biological processes including spermatogenesis, hind limb formation, hematopoiesis, and immune regulation Figure . PLZF acPLZF expression is lost in many cancer cells (Figure PLZF expression has been correlated to many malignant phenotypes in different cancer cell types, including increased proliferation, enhanced invasiveness and motility, and resistance to apoptosis. All of these features are required for cancer cell survival, therefore PLZF is considered to be a tumor suppressor gene. Thus the requirement to turn off the expression of PLZF for tumorigenesis is evident, but the exact mechanism has yet to be elucidated. Possible mechanisms include epigenetic modifications or up-regulation of other transcription factors in order to silence the unfavorable PLZF expression, microRNAs to block mRNA translation, or signals to reduce the stability of the PLZF protein.Many biological functions of PLZF in differentiated cells remain to be determined. The number of regulatory factors controlled by PLZF or by its target genes is substantial. Although expressed in many tissue types, its expression level is low compared to the levels in stem cells or progenitor cells. However, this does not mean that it has a limited function in differentiated cells, as can be seen by the large cohort of biological functions listed in Table s Figure . As discPromyelocytic leukemia zinc finger can also regulate the expression of different microRNA molecules. This ability is important, because it can aid in the understanding of how PLZF achieves the transcriptional repression of many downstream targets. This also adds an extra level of regulation to the already known promoter-dependent transcriptional regulatory activities of PLZF. A single microRNA molecule not only can target a single pre-mature mRNA, but also can regulate a plethora of mRNA molecules, leading to an amplified transcriptional regulatory signal that could affect the basic properties of the cell, including proliferation rate, apoptosis resistance, migration, and invasiveness.Understanding the biological activities of PLZF and its DNA/protein/miR interaction network would augment our understanding of its complex involvement in many pathways and may delineate the potential of fine tuning its expression profile in cancer cells. This would add to its value as a therapeutic target to modulate malignant cellular characteristics in oncological disease backgrounds.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Many HIV/AIDS patients experience pain often due to advanced HIV/AIDS infection and side effects of treatment. In sub-Saharan Africa, pain management for people with HIV/AIDS is suboptimal. With survival extended as a direct consequence of improved access to antiretroviral therapy, the prevalence of HIV/AIDS related pain is increasing. As most care is provided at home, the management of pain requires patient and family involvement. Pain education is an important aspect in the management of pain in HIV/AIDS patients. Studies of the effectiveness of pain education interventions for people with HIV/AIDS have been conducted almost exclusively in western countries.A randomised controlled trial is being conducted at the HIV and palliative care clinics of two public hospitals in Malawi. To be eligible, patient participants must have a diagnosis of HIV/AIDS (stage III or IV). Carer participants must be the individual most involved in the patient’s unpaid care. Eligible participants are randomised to either: (1) a 30-minute face-to-face educational intervention covering pain assessment and management, augmented by a leaflet and follow-up telephone call at two weeks; or (2) usual care. Those allocated to the usual care group receive the educational intervention after follow-up assessments have been conducted (wait-list control group). The primary outcome is pain severity measured by the Brief Pain Inventory. Secondary outcomes are pain interference, patient knowledge of pain management, patient quality of life, carer knowledge of pain management, caregiver motivation and carer quality of life. Follow-up assessments are conducted eight weeks after randomisation by palliative care nurses blind to allocation.This randomised controlled trial conducted in sub-Saharan Africa among people living with HIV/AIDS and their carers will assess whether a pain education intervention is effective in reducing pain and improving pain management, quality of life and carer motivation.ISRCTN72861423.Current Controlled Trials  It is estimated that 34 million people were living with HIV/AIDS at the end of 2010 [Sub-Saharan Africa has 10% of the world’s population, but it is home to 67% of all people living with HIV/AIDS, making it the region worst-affected by HIV/AIDS . Antiretth worst-affected country with HIV/AIDS worldwide [In Malawi the prevalence of HIV/AIDS is estimated at 11% of the population aged between 15 and 49 years, with around 920,000 people living with HIV/AIDS at the end of 2010 [orldwide . One iniAdvanced HIV disease infection and its treatment with HIV medication are associated with physical and psychological symptoms. These require focused assessment and management using locally available resources and interventions to optimise quality of life for patients and their carers . The negInadequate pain control remains a challenge for HIV/AIDS patients and has an impact on their quality of life ,20. PainThe aim of this trial is to evaluate the effect of an educational intervention for patients with HIV/AIDS and their carers. The study will test the following hypotheses:1. Compared with usual care, patients with HIV/AIDS who receive a pain education intervention will report less severity of pain.2. Compared with usual care, patients with HIV/AIDS who receive a pain education intervention will report less interference of pain in their daily activities.3. Compared with usual care, patients with HIV/AIDS who receive a pain education intervention will have a greater knowledge of pain management.4. Compared with usual care, patients with HIV/AIDS who receive a pain education intervention will have a better quality of life.5. Compared with usual care, carers of patients with HIV/AIDS who receive the pain education intervention will have greater knowledge of pain management.6. Compared with usual care, carers of patients with HIV/AIDS who receive the pain education intervention will have greater motivation to provide care.7. Compared with usual care, carers of patients with HIV/AIDS who receive the pain education intervention will have a better quality of life.The study is a two-centre randomised wait-list controlled trial. Participants (patients with HIV/AIDS and their carers) randomly allocated to a pain education intervention group receive a leaflet-based educational intervention and verbal instructions for approximately 30 minutes on pain assessment and management in addition to usual care. Participants randomly allocated to the usual care group receive standard care, but receive the leaflet-based educational intervention on completion of follow-up measures for both treatment groups (wait-list control). Participants are assessed at baseline after providing informed consent and then randomly allocated to either the pain education intervention or usual care arm of the trial. Follow-up assessments are conducted after eight weeks.The trial setting is that of HIV and palliative care clinics within two public hospitals in northern Malawi. Both hospitals  provide in-patient, clinic-based and home-based care for people with HIV/AIDS that includes active treatment and palliative care. Ekwendeni Hospital provides services funded by the government. It was one of the first hospitals in Malawi to provide free HIV/AIDS medication. Mzuzu Central Hospital is government-funded and the largest referral hospital in north Malawi for people with HIV/AIDS. The population served by these hospitals includes people from both rural and urban areas.Participants are people living with HIV/AIDS and their carers. All participants need to be able to read and write in English or Tumbuka . They must be adults aged 18 years or over.To be eligible for the trial, participants must have received a diagnosis of HIV/AIDS. Participants with other conditions, such as cancer and tuberculosis, are included if these conditions present alongside a diagnosis of HIV/AIDS. Eligible participants with HIV/AIDS must be at WHO clinical stages III or IV of HIV/AIDS, or with a CD4 cell count of less than 350 cells, when the presence of pain and other symptoms are more likely due to opportunistic infections or side effects of HIV treatment. Staging for trial eligibility is assessed from the medical records if recorded or through assessment by clinic staff if this information is not available.To be eligible for inclusion, carers must be living with the person with HIV/AIDS and be identified as the individual most involved in their care.People living with HIV/AIDS will be excluded if they have health problems that may hinder cognition and communication, such as HIV-associated dementia. This is assessed by the attending clinical officer during history-taking at the initial assessment or at clinic review.People living with HIV/AIDS in Malawi typically visit the hospital  with their family members. Posters about the study entitled ‘Pain Education Study’ are prominently displayed and potential participants have the opportunity to be given further information about the study directly from KN or from clinic staff.The study is introduced either during the first appointment at the HIV clinic for newly registered patients or during routine appointments at the HIV clinics or palliative care clinics for those who are already receiving HIV medication  and demographics. Other measures taken at baseline are those used as outcomes for the trial.The primary outcome is pain severity measured using the Brief Pain Inventory . A range1. Pain severity is measured using the single item of the Brief Pain Inventory (BPI-PS)  where pa2. Pain interference with daily activities is measured using the mean score of the seven pain interference items of the Brief Pain Inventory (BPI-PI). These items measure, on a scale of 0 to 10, the degree to which the patient reports pain interfering with each of seven activities  and is the recommended method of assessment of pain-related functional impairment in clinical trials .3. For patients, knowledge of pain management is measured using the knowledge subscale of the Patient Pain Questionnaire (PPQ-K) . The PPQ4. For patients, quality of life is measured using the APCA African POS . The APC5. For carers, knowledge of pain management is measured using the knowledge subscale of the Family Pain Questionnaire (FPQ-K) . Like th6. Carer motivation is measured using the Picot Caregiver Rewards Scale (PCRS) . The PCR7. For carers, quality of life is measured using the APCA African POS . The APCWhile the BPI  and APCAFollow-up measures are conducted after eight weeks following delivery of the intervention. Nurses blind to treatment group conduct the follow-up assessments. This is implemented during the routine appointments to the HIV or palliative care clinic.P-value cut-off of 0.05 to determine a statistically significant result, 76 people per arm of the trial will be needed to complete the study to give 80% power to detect such a difference. This is based on a review [We wish to be able to detect a mean difference of 10% between the treatment groups in the primary outcome measure (average pain severity in the BPI). A 10% improvement is the lower limit of changes considered clinically important . Using aa review  that sugWe will provide a descriptive account of the two treatment groups at baseline in terms of demographics, recruiting centre, stage of HIV/AIDS and baseline values of all study outcomes. All patients and carers will be analysed according to the group to which they were randomised. Treatment groups will be compared in terms of our primary outcome measure (pain severity using the BPI-PS treated as a continuous measure) using a linear regression model with baseline BPI and treatment group and recruiting centre as covariates. Analysis of each of the six secondary outcomes  will be conducted using six equivalent models with estimates of treatment effect conditional on the value of the outcome at baseline. Sensitivity analysis will be reported and performed as follows: we will conduct secondary analyses that (1) adjust for variables that are potential predictors of outcome  and (2) make worst-case and best-case scenario assumptions about participants lost to follow-up using the Stata command ‘rctmiss’ . AnalysiThe study has been approved by the University of Nottingham Medical School Research Ethics Committee (SNMP 11042012) and National Health Sciences Research Committee of Malawi (NHSRC 1023).Findings from this trial will inform the management of pain experienced by people living with HIV/AIDS. Previous trials of interventions designed to enhance self-management for people living with HIV/AIDS have been conducted either exclusively ,32,50 orThe trial commenced recruiting in September 2012. We anticipate reaching our recruitment target by June 2013.AIDS: Acquired immunodeficiency syndrome; APCA: African palliative care association; ART: Antiretroviral therapy; BPI: Brief pain inventory; FPQ: Family pain questionnaire; PCRS: Picot caregiver rewards scale; POS: Palliative care outcomes scale; PPQ: Patient pain questionnaire; RCT: Randomised controlled trial; UNAIDS: United Nations Program of HIV and AIDS; WHO: World Health Organisation.The authors declare that they have no competing interests.KN, JS and AA were responsible for the development and refinement of the protocol. KN is the principal investigator and trial manager. KN will conduct data analysis under the supervision of AA. KN and AA wrote the initial draft of the manuscript. KN, JS and AA contributed to, edited and approved the final manuscript.


The aim of this study was to determine the prevalence and clinical significance of c-MET expression in hormone-naïve prostate cancers. A pre-existing prostate tissue microarray (TMA) containing samples of 4,177 patients treated by radical prostatectomy was used. A total of 3,378 different prostate cancers were successfully analyzed for c-MET expression by immunohistochemistry and follow-up data were available for 4,104 patients. Membranous c-MET immunostaining was performed for 2,655 (78.6%) tumors. High c-MET protein expression was significantly associated with a high Gleason grade (P=0.0018). However, c-MET was not a prognostic marker for biochemical recurrence. c-MET levels were also not associated with other parameters, including tumor stage, nodal stage and surgical margin status. The c-MET protein is often overexpressed in prostate cancer, but has no prognostic relevance. However, the frequent presence of high levels of membranous c-MET protein in prostate cancer cells makes c-MET an attractive target for imaging and treatment. The receptor protein kinase MET was originally identified as an activated oncogene , with heMET expression occurs in the normal prostate epithelium. In contrast to luminal cells, basal cells show consistently high c-MET expression . Data onThe aim of this study was to clarify the prevalence and prognostic role of c-MET expression in prostate cancer by using a pre-existing tissue microarray (TMA) including more than 4,000 prostate cancers, the majority with clinical follow-up data. The data show an abundant expression of c-MET and demonstrate that c-MET protein analysis does not serve as a prognostic marker for prostate cancer patients.A pre-existing prostate cancer TMA consisting of tissue samples from radical prostatectomy specimens of 4,177 patients, consecutively treated at the Department of Urology and the Martini Clinic at the University Medical Center Hamburg-Eppendorf  between 1997 and 2008 , was useFreshly cut TMA sections were stained on 1 day in a single experiment. High-temperature pre-treatment of slides was performed in an autoclave in citrate buffer, pH 7.8 for 5 min. c-MET immunostaining was performed using a monoclonal antibody . The Envision system  was used to visualize the immunostaining. Only membranous staining was evaluated as cytoplasmatic staining, if present, was always linked with stronger membranous staining. The staining intensity  and the fraction of positive tumor cells were recorded for each tissue sample. A final score was created from these 2 parameters according to the following scores: negative scores had a staining intensity of 0; weak scores had a staining intensity of 1+ in ≤70% of tumor cells or a staining intensity of 2+ in ≤30% of tumor cells; moderate scores had a staining intensity of 1+ in >70% of tumor cells or a staining intensity of 2+ in >30% but ≤70% of tumor cells or a staining intensity of 3+ in ≤30% of tumor cells; and strong scores had a staining intensity of 2+ in >70% of tumor cells or a staining intensity of 3+ in >30% of tumor cells.For statistical analysis, the JMP 8.0 software  was used. Contingency tables were calculated to determine the association between the c-MET immunostaining score and clinicopathological variables. The Chi-square test was used to identify significant associations. Kaplan-Meier curves were generated for PSA recurrence-free survival. The log-rank test was performed to determine the significance of differences between stratified survival functions. Cox proportional hazards regression analysis was performed to determine the statistical independence and significance between pathological, molecular and clinical variables.A total of 3,378 (81.6%) tumor samples were successfully analyzed in our TMA analysis. The reasons for non-informative cases  included a lack of tissue samples or absence of unequivocal cancer tissue in individual TMA samples.c-MET immunostaining revealed strong membrane staining in our tissues. Although certain cytoplasmic staining was sometimes observed, this was always associated with a markedly higher staining level at the membranes. Membranous c-MET immunostaining was recorded in 2,655 (78.6%) of 3,378 successfully analyzed cases. Staining was weak in 780 (23.1%), moderate in 801 (23.7%) and strong in 1074 (31.8%) prostate cancers . In beniet al of 3,378 successfully analyzed cancers and strong c-MET expression was significantly associated with a high Gleason grade (P=0.0018). These results are within the range of previous studies using immunohistochemistry, although the reported c-MET expression levels vary from 33 to 84% ,13–15. Het al. Watanab al(et al investiget al(et al required only more than 5% to classify a tumor as positive (et al(et al(et al(It is possible that these controversial data are partly attributable to sampling issues as these studies have all analyzed markedly small patient cohorts and are characterized by highly variable definitions of c-MET-positivity. Watanabe et al considerpositive . Knudsenve (et al evaluate al(et al, secreto al(et al,14. In c al(et al did not c-MET is frequently expressed in a variety of other cancers. For some cancers, including cholangiocarcinoma, gastric or skin cancer, a clear correlation between c-MET expression level and a poor prognosis has been demonstrated ,21,22. AThe high frequency of expression in prostate and other cancer types makes c-MET an attractive potential therapeutic target. Recently, several studies with c-MET inhibitors were realized or are in progress ,25. ReceIn conclusion, the results of this study reveal that c-MET is frequently overexpressed in prostate cancer. A significant correlation was demonstrated between strong c-MET expression and high Gleason grade, but not with other clinicopathological parameters. Although c-MET appears to be involved in the progression of prostate cancer, this study does not confirm a role of c-MET as a prognostic marker in patients with prostate cancer.


Cancers of the hepatobiliary tract  are characterized by relatively infrequent occurrence, aggressive growth, and recurrences after treatment. Hepatocellular carcinoma (HCC) is of special concern in the context of HIV/AIDS due to substantial associated morbidity and mortality. The overall burden of liver cancer may increase in people with AIDS as the combined effects of alcohol use, coinfection with hepatitis C virus (HCV) and hepatitis B virus (HBV), and other risk factors manifest as chronic liver disease.Registry linkage data from the U.S. HIV/AIDS Cancer Match Study were used to estimate standardized incidence ratios (SIRs) to compare the risk of hepatobiliary cancers in people with HIV/AIDS to the general population. We also estimated rate ratios (RRs) of HCC by HIV risk group, calendar period and AIDS status. HIV risk groups were categorized by HCV prevalence .Compared to the general population, people with AIDS had higher risk for HCC . SIRs were also elevated for other liver and intrahepatic bile duct tumors , but not for cholangiocarcinomas  or gallbladder tumors . Adjusted for sex and age, people with high HCV prevalence were at higher risk for HCC than people with lower HCV prevalence . SIRs were elevated for all HIV risk groups, with the highest SIRs among hemophiliacs (40.4), IDUs (5.59), and IDU-MSM (4.39). Steadily increasing risk among persons with AIDS was observed across calendar time, including during the HAART era (Figure This study reinforces the primary role of HCV coinfection in HCC pathogenesis in persons with AIDS in the United States. HCC risk is higher in people with AIDS than people with HIV infection without AIDS, consistent with a contribution from prolonged immunosuppression. Rising HCC incidence in the era of HAART suggests that HAART itself does not fully correct the negative impact of HIV on HCV-related cirrhosis, and that access to appropriate anti-HCV therapies in HIV-infected individuals is critical for prevention of progression to HCC.