To investigate efficacy and safety of ultrasonography-guided local corticosteroid and anesthetic injection followed by physical therapy for the management of quadriceps fat pad (QFP) edema.We prospectively evaluated 1671 knee MRI examinations in 1542 patients for QFP edema with mass effect, which was present in 109 (6.5%) knees. Participants were assigned into injection and therapy groups . Injection group was first treated with ultrasonography-guided QFP injection of 1 mL corticosteroid and 1 mL local anesthetic agent. Patients were evaluated at baseline and 1-, 2-, 6-month follow-up for pain using static and dynamic visual analogue scale (VAS), suprapatellar tenderness, and QFP edema on MRI.static-dynamic VAS scoring, respectively), whereas there was no such superiority at the sixth month. No severe adverse events were identified.Final sample size consisted of 19 knees  in 17 patients. An overall improvement was detected in both groups between baseline and final assessments. The injection group fared better than the therapy group in static VAS scores (3.33 ± 1.70 versus 0.56 ± 1.33), while there was no such difference for dynamic VAS. Incidence of suprapatellar tenderness decreased in both groups, statistically significantly in the injection group (from 100% to 0%). Pain reduction was greater in the injection group at the first month (88.9% – 90% good response versus 50% – 66.7% good response, Ultrasonography-guided local injection followed by physical therapy is safe in the management of QFP edema; however, it is not superior to stand-alone physical therapy program in the long term. The quadriceps (suprapatellar) fat pad (QFP) is an extrasynovial structure bordered anteriorly by the quadriceps tendon and posteriorly by the suprapatellar recess of the knee joint . QFP ede2Several studies have explored the frequency and MRI characteristics of QFP edema and/or the mass effect, and their relationships to knee pain and structural abnormalities of the knee 34. Quadr3232342346The aim of the present study was to explore the efficacy and safety of ultrasonography (US)-guided local corticosteroid and anesthetic injection, followed by physical therapy, to manage QFP edema. We present the preliminary report of the 6-month follow-up data.This prospective, consecutive-enrollment, non-randomized, two-center , controlled clinical study was performed with approval of our institutional ethical review board, and written informed consent was obtained from all patients from both centers. Two patients with anterior knee pain and QFP edema with mass effect on MRI  first underwent US-guided QFP injection as described below in the first center.We then prospectively evaluated in the second center 1,671 consecutive knee MRI examinations of 1,542 patients with various knee symptoms imaged between December 2011 and September 2014. A total of 109 knees (6.5%) of 94 patients (6.0%) exhibited QFP edema with a mass effect. A total number of 23 potentially eligible patients (24.5% of those with QFP edema displaying mass effect on MRI) with anterior knee pain were examined by a single orthopedic surgeon with four years of experience. A total number of 19 patients (20.2%) who met the clinical and radiological criteria were recruited for the study. However, one patient refused to participate, and the study commenced with the remaining 20 patients . A total number of 18 patients (20 knees) were available for the 6-month follow-up; two patients  were lost to follow-up , the presence of anterior knee pain, suprapatellar tenderness , and an age of 18 years or over. The exclusion criteria included pregnancy or lactation, a history of knee injury in the prior year, open or arthroscopic knee surgery in the prior year, any previous diagnostic or therapeutic intra-articular injection, a congenital or developmental disorder that could have disrupted knee shape or alignment, regular use of oral analgesics in the past 10 days and regular physical therapy in the three months prior to the current knee MRI. Other exclusion criteria were extensive lymphedema around the knee, a partial or full-thickness anterior or posterior cruciate ligament tear, a full-thickness collateral ligament or retinaculum tear, any displaced tear of the menisci or any loose intra-articular body.All knee MRI examinations were performed on 1.5 T scanners  using dedicated knee coils. Each patient was placed supine on the MRI examination table with the knee at 10˚–15˚ of flexion. Our standard knee MRI protocol consisted of the following sequences: sagittal fat-suppressed (FS) proton density-weighted (PDW) fast spin echo (FSE), FS T2-weighted (T2W) FSE, and T1W FSE; coronal FS T2W FSE; and axial FS PDW FSE. On follow-up visits (see below), we ran only three sequences of the standard knee MRI protocol: sagittal FS PDW, FS T2W, and T1W.MRI examinations of 18 patients were reviewed by the same musculoskeletal radiologist (blinded) with two years of experience in musculoskeletal imaging. MRI of the remaining two patients have been evaluated by a senior musculoskeletal radiologist (blinded) with 17 years of experience in musculoskeletal MR imaging at the first center in which the pilot study of the QFP injection was conducted and physical therapy regime was designed.  We considered by visual estimation that QFP edema was present when the signal intensity of at least two-thirds of the fat pad was greater than that of the surrounding normal muscle tissues on sagittal FS PDW or FS T2W images along with a mass effect – that was evident with a convex posterior fat-pad border on sagittal MR images Figure .Patients were divided into two groups by reference to their treatment preferences. The therapy group included those who refused local injection, and the local injection group those who consented to injections of local anesthetic and steroid into the fat pad.The same orthopedic surgeon examined 18 participants within five days following routine knee MRI to determine whether point tenderness was evident over the suprapatellar region . The remaining two patients were examined similarly by a senior orthopedic surgeon (with 18 years of experience) at the pilot center.All study participants attended a baseline session (Day 0) which included the following: collection of demographic information (age and gender); routine knee MRI; and scoring of pain on a 10-point visual analog scale (VAS)  in a neutral (supine with the knee at 0° flexion) position (static VAS), and when standing up after having been in a seated or squatting position (dynamic VAS). Ultrasonography-guided injections Figure  were perA total of 2 mL [1 mL bupivacaine hydrochloride  and 1 mL methylprednisolone ] was injected into the fat pad of each consenting patient within five days of routine knee MRI after their pain scoring at the baseline session. All injections were administered with the patient supine and the knee under slight flexion . Following administration of 3–5 mL prilocaine hydrochloride  to establish local anesthesia, we advanced a 22-gauge needle obliquely, using an anteromedial approach, under ultrasonography control as described by Van Le and Harish . We recoAt 1-month follow-up (Day 30), the following procedures were performed: limited knee MRI (see the three sequences below); physical examination of the knee, including assessment of suprapatellar tenderness, by the same respective orthopedic surgeon who had performed the initial physical examination at study centers; static and dynamic VAS scoring; and recording of adverse events. The physical therapy program continued. The same procedures were repeated at the 2-month (Day 60) and 6-month follow-up (Day 180) Figure .The primary efficacy end-point was clinical improvement, as evaluated by changes in VAS scores from baseline to any follow-up visit after injection. The secondary efficacy parameters included between-group and intra-group variations in VAS scores, suprapatellar tenderness, and the extent of QFP edema with a mass effect evident on MRI at follow-up visits. In addition, we evaluated pain reduction by comparing differences between baseline and follow-up VAS scores. The responses to treatment were classified using the scheme of a recent retrospective study assessing the effectiveness of US-guided corticosteroid injection into the quadratus femoris muscle to treat patients with ischiofemoral impingement : good ret-test was used to compare differences in age and anthropometric measures between the groups, and the Mann-Whitney U test was employed to compare VAS scores. Nominal data were analyzed using Fisher’s exact test. We employed the McNemar or Wilcoxon signed rank test, as appropriate, to determine whether differences evident between any two follow-up visits were significant. A p value less than 0.05 was considered to reflect statistical significance. The Bonferroni correction was applied to minimize Type 1 error upon all multiple comparisons .Data analysis was performed using SPSS for Windows, version 11.5 . Data are shown as means ± standard deviations (SDs), or as numbers of cases with percentages, as appropriate. Student’s We recruited 10 (55.5%) males and 8 (44.4%) females . Follow-up data were collected from all 18, but we excluded one male patient from analysis when he disclosed non-compliance with the physical therapy program  were significantly higher in the injected than in the therapy group, but neither gender nor knee sidedness differed. The demographic data are summarized in Table p = 0.003)    Table . The mea) Figure . Moreove) Figure .p > 0.0125, after Bonferroni correction) (Table p = 0.004).The dynamic VAS scores were similar in both groups at baseline (n) Table . Howevergood scores at the 1- and 2-month follow-ups, and 77.8% at the 6-month follow-up; the respective figures for the therapy group were 50% and 100%). In terms of changes in dynamic VAS scores, the injected group reported more relief at the 1-month follow-up, but the scores of both groups were the same at the 2- and 6-month follow-ups   Table .p = 0.002 and p = 0.002, respectively), but no significant between-visit difference was evident in the therapy group.This incidence decreased in both groups, particularly the injected group, at the time of the final (6-month) assessment, compared to the baseline values Figure . The decThe incidence of QFP edema on MRI did not significantly differ between or within the two groups throughout the study period Figure . The incThe treatments were tolerated well and no severe adverse event was noted; however, almost all patients  in the injected group reported markedly increased local pain  several hours after intra-fat pad injection that disappeared within 1–2 days.In this prospective, controlled preliminary clinical study to assess the efficacy and safety of US-guided intra-fat pad injection in symptomatic patients with QFP edema, we found clinical improvements in both the injection and therapy groups after six months of treatment. Pain reduction was greater in the injected group at the 1-month, but not at the 6-month, follow-up. Thus, US-guided local injection followed by physical therapy is an effective and reliable treatment for QFP edema, and injection affords more effective short-term pain reduction. However, local injection is no better than stand-alone physical therapy in the longer term.steroid injections into four patients eliminated all symptoms, with no recurrence; however, the details of the injections and follow-up duration were not given [Few data on the management of patients with QFP edema and a mass effect are available. Previous articles discussed the MRI characteristics of such patients and relationships thereof with particular structural abnormalities of the knee 345. Roth34ot given . Additioot given .To date, no report has detailed the nature and amount of medication given, the injection method, or clinical follow-up data on QFP edema, with the exception of one case report . We studAlthough injections afforded rapid relief, consistent with the established efficacy of corticosteroids for reducing inflammation and relieving pain in the early stages of a wide array of inflammatory conditions, physical therapy alone in our study was associated with gradual clinical improvement over time. Restoration of patellar tracking biomechanics by active (quadriceps retraining) and passive  exercises improves lower limb control and patellar congruence of the joint, contributing to pain improvement . Thus, lMRI is the most useful tool for diagnosis of soft tissue abnormalities such as QFP edema with a mass effect. Several previous studies have yielded consistent estimates of the prevalence of the condition where only QFP mass effect, but not edema-like signal, was assessed. This was 12% (11/92 knee MRI examinations) in the work of Roth et al. ; 13.8%  has been suggested to trigger OFP enlargement, particularly in subjects with bilateral clinical and radiological findings . However2233Our study has several limitations. First, the statistical power is low because of our relatively small sample size. Our ability to detect significant changes was thus reduced. Second, non-randomized design of the study caused patient selection bias. Third, the between-group differences in BMI and age might have increased the error variance and reduced the ability to detect small differences. Fourth, the higher baseline static VAS scores of the injected group and the possibility of lower-than-full-compliance with the physical therapy regimen  might have somewhat compromised our ability to detect the true effects of treatment, and differences between the study groups. We did, however, specifically question at follow up visits the patients’ compliance with the physical therapy regimen and excluded from the study one patient who disclosed none compliance. Fifth, we did not attempt to exclude patients with patellofemoral compartment cartilage degeneration, which may be encountered in persons at the age groups as in our study and might have been the cause of (or at least have contributed to) anterior knee pain; this condition (seen in two of our cases) by itself might have benefited from our treatment protocol. Although the lack of a placebo control group may be considered as yet another limitation, placebo injections in humans are not acceptable according to our institutional review board’s code of conduct. Finally, we did not assess patients for longer than six months. Therefore, whether the effect of local drug injection extends beyond this time is unknown.Despite these limitations, the results of our controlled preliminary clinical study show that US-guided local injection followed by physical therapy is effective and well-tolerated when used to manage QFP edema. Injection is effective in terms of pain reduction in the near-term. However, local injection is not superior to physical therapy alone in the longer term.


Hepatitis D virus (HDV) infection leads to the most severe form of chronic viral hepatitis and requires the attention of a liver specialist. In this review, I will recapitulate recent advances in the management of HDV, present background information on HDV infection as well as current chronic hepatitis D treatment, briefly examine the HDV life cycle and discuss new management strategies. Only as a dual infection with HBV can HDV cause liver disease in humans. The helper function needed by HDV from HBV is hepatitis B surface antigen (HBsAg) synthesis. HDV needs HBsAg for virion assembly when the HDV nucleoprotein complex, formed in the nucleus and secreted into the cytoplasm, is covered by HBsAg to complete virion morphogenesis. The HBsAg envelop of the HDV virion is critical for cellular attachment and for the propagation of HDV to other hepatocytes3.Hepatitis D virus (HDV) infection leads to the most severe form of chronic viral hepatitis and needs attention from a liver specialist in this context. Since its discovery in the 1970s by Mario Rizzetto and co-workersThe aim of this review is to recapitulate recent advances in the management of HDV. For this, some background information for HDV infection in men, including current treatment of chronic hepatitis D (CHD), will be provided. New management strategies will be preceded by a short debrief of the HDV life cycle.4 and prospectively in intravenous drug addicts in men5. Otherwise, acute HDV infection cannot be differentiated from other acute hepatotropic viral infections on clinical grounds. However, HDV infection may more often be associated with a severe course, and frequent occurrence of fulminant delta hepatitis has been reported both in the US and in Europe7. However, in recent years, fulminant delta hepatitis is less frequently encountered8, which may be linked to the slower turnover of HDV in the community9. The chronicity rate of HBV–HDV co-infection in adults appears to be no different than the chronicity development rate after acute hepatitis B mono-infection10.HDV leads to liver disease in humans either as co-infection with HBV or as superinfection of a chronic HBsAg carrier with HDV. The consequence of the former scenario is acute HDV infection. Acute HDV hepatitis may be associated with a biphasic clinical presentation, possibly related to sequential expression of the two viruses, observed both in early chimpanzee studies12. However, the severity of the course of CHD may be dependent on host and viral factors, as CHD is an immune-mediated disease13. Among viral factors, both HDV and HBV genotypes matter. Genotype II HDV seen in the Far East is associated with a milder form of chronic hepatitis compared to genotype I, which has a worldwide distribution15. Genotype III HDV is observed in the Amazon region of South America and is associated with a particular severe clinical presentation16. Genotype IV, reported formerly as genotype IIb, has been seen both in the Far East (Japan and Taiwan) and in Africa19. Genotypes V to VIII are seen exclusively in Africa20. African CHD may be associated with a mild form of the disease and may respond better to interferon (IFN) treatment, as has been reported very recently21.CHD is associated with the most severe form of hepatotropic-induced viral hepatitis22. In a study from Taiwan, HBV genotype C was linked to more severe disease compared to genotype B. Multivariate analysis identified age, HBV genotype C, and HDV genotype I as independent factors for poor outcomes14.In addition to HDV genotypes, HBV genotypes may also contribute to the clinical presentation of CHD. In a study from Brazil, HDV viral load was reported to be lower in genotype A compared to genotype D or F patients23. Therefore, in non-endemic areas, special attention to high-risk patients for acquiring HDV is needed. These are mainly intravenous drug users and immigrants from HDV-endemic countries, which may represent 55 to 95% of the total HDV population in Western European countries including Germany, Italy, Spain, Sweden, France, and England31. Endemic countries or regions for HDV include Mongolia, Pakistan, sub-Saharan Africa, Turkey, Romania, Albania, former Soviet republics, and the Amazon region in the northern part of South America32.Delta hepatitis has received the designation of orphan disease both in the US and in the European Union. This emphasizes that in both geographical regions, HDV infection is infrequently encountered. In such circumstances, it is not easy to follow expert advice to test every HBsAg carrier for HDV, which involves testing a cheap serological marker. However, forgetting to test may have dreadful consequences for patients. This is highlighted in a recent study from the US where, out of 1,191 chronic hepatitis B (CHB) patients, only 499 had been tested for anti HDV. Out of these 499 patients, 42 (8%) were anti HDV positive and among HDV co-infected patients 70% had cirrhosis33. Combination treatment of peg-IFN with lamivudine35, ribavirin37, adefovir38, and tenofovir39 was explored but was disappointing, as it did not lead to better viral response rates compared to IFN monotherapy. The only exception was peg-IFN–adefovir combination therapy, which was also not associated with higher viral response rates compared to peg-IFN monotherapy but interestingly the decline of quantitative HBsAg levels was more pronounced compared to peg-IFN monotherapy38. It was hoped that a similar effect on HBsAg levels would also be seen in the HIDIT-2 study, which compared peg-IFN–tenofovir combination with peg-IFN alone, but no such effect was observed39.The management of CHD has not changed in over 30 years and consists of treatment with IFNs. The only modification in therapy is the switch from conventional to pegylated IFN (peg-IFN), which probably has not led to better viral response rates but merely has the comfort for patients of once-weekly dosing compared to the thrice-weekly schedule of conventional IFNs, thanks to the more suitable pharmacodynamic properties of peg-IFNs. Peg-IFN alpha 2a should be administered as subcutaneous injections at a dose of 180 μg once weekly and peg-IFN alpha 2b at a dose of 1.5 μg/kg, similar to the dosing schedule used before the era of directing acting antiviral in hepatitis C infection. The optimal duration of treatment is not known, but treatment for 1 year is the most-studied and -used treatment duration33. This can be attributed to the inefficacy of NAs on HBsAg levels, as production of HBsAg represents the only HBV function needed by HDV. In contrast, a median 6 years of tenofovir treatment was effective in HIV–HDV co-infected patients40. The exact mechanism of efficacy of tenofovir in HIV–HDV co-infected patients is not clear but needs explanation. Longer duration of NA treatment may lead to decreased recycling of HBV DNA to the nucleus, thus decreasing the HBV covalently closed circular DNA (HBV ccc DNA) pool with consequent decreases in HBsAg levels41. However, another explanation could be that the observed beneficial effect may have been a result of immune re-constitution secondary to effective antiretroviral therapy42. Further studies would be helpful. One such study has to deal with prolonged NA treatment in CHD without HIV infection. Since prolonged NA treatment has been reported to lead to a decrease in quantitative HBsAg levels43, this deserves to be explored.Treatment with nucleoside/nucleotide analogs (NAs), currently the treatment of choice for CHB, was ineffective in CHD where NAs were tested for a period of 6–12 months44. First, heparan sulfate proteoglycans on the hepatocyte membrane are approached by the HDV virion46 followed by attachment to the entry receptor sodium taurocholate co-transporting polypeptide (NTCP)47. At this stage, the integrity of the amino acids in the pre-S1 region is crucial. Once inside the hepatocyte, HDV is uncoated and the ribonucleoprotein complex is directed to the nucleus where HDV replication occurs through a double rolling circle mechanism48. Importantly, RNA polymerase needed during replication is a host enzyme, making it an unsuitable target for drug intervention, contrary to the hepatitis B or C viruses, which use their own polymerase product12. HDV RNA encodes for one protein, the small hepatitis D antigen (S-HDAg), which promotes HDV RNA replication. Another protein, large HDAg (L-HDAg), is synthesized as a result of an editing event, catalyzed by the host enzyme adenosine deaminase acting on double-stranded RNA (ADAR1)49. L-HDAg inhibits HDV RNA replication but is crucial for virion assembly12. The newly formed ribonucleoprotein complex is exported to the cytoplasm and is coated with the HBV envelop covering the L-, M-, and S-domains of HBsAg. The nucleoprotein complex HBsAg bond is possible through a cysteine-containing 4-amino-acid motif at the L-HDAg, which serves as a substrate for prenyltransferases. There are two prenyltransferases, farnesyltransferase and geranylgeranyltransferase. Of these two enzymes, farnesyltransferase is used by HDV. Through the latter enzyme, a 15-carbon farnesyl moiety is attached to the large delta antigen35. The HBsAg cover of the ribonucleoprotein complex is a prerequisite for successful completion of virion morphogenesis in HDV50. The new virion then exits the cell either via multivesicular bodies or by utilizing the subviral particle secretion pathway via the Golgi44.HDV enters the hepatocyte after attachment of the HDV virion to the hepatocyte. Entry into the hepatocyte is similar to the entry of HBV, as both HBV and HDV cellular attachment is dependent on various common structures within the HBsAg where the pre-S1 domain of the L protein plays a key role51 would be beneficial in CHD as well. After all, the ideal surrogate marker for effective treatment is HBsAg clearance, and the reason we use quantitative HDV RNA testing as a surrogate in daily practice and not HBsAg clearance is that the latter is so rarely achieved. However, patients who have a lasting virologic response to treatment have a high chance of clearing HBsAg on long-term follow-up. In a study from our own institution, we were able to show that 37% of patients with virologic response to conventional or peg-IFN lost HBsAg during a median follow-up of 5 years52.In the last 5 years, new drugs have been explored for the management of CHD. These drugs tailor various steps in the life cycle of HDV. However, as a general comment, it is clear that any management strategy leading to a functional or complete cure in HBVNew compounds currently explored with relevance to the management of CHD are depicted in53. Myrcludex B was tested both in intravenous and in subcutaneous formulations  or 6 months of Myrcludex B plus peg-IFN followed by 6 months of peg-IFN. Myrcludex B led to a >1 log10 reduction in HDV RNA in 6 out of 7 patients at the end of 24 weeks’ treatment56. One patient became HDV RNA negative on monotherapy with Myrcludex B. Combination of Myrcludex B with peg-IFN was more effective, and 5 out of 7 patients became HDV RNA negative. The delta decline in HDV RNA was 1.67, 2.17, and 2.59 log10 with Myrcludex B, peg-IFN monotherapies, and their combination, respectively. HDV RNA negativity with combination treatment in 5 out of 7 patients after 6 months of treatment is promising, and a synergistic effect for combination treatment has been suggested56. The primary endpoint of the study, a decrease in HBsAg levels of 0.5 IU/mL at any time during the study56, was not reached, however. Further studies with Myrcludex B with different dosing and treatment duration regimens are to be expected, and such a study is already ongoing.Myrcludex B, a myristoylated lipopeptide comprising 47 amino acids of the pre-S1-domain of the HBV L-surface protein, represents the first hepatocyte entry inhibitor. As pointed out earlier, myristoylation of glycine in the pre-S1-domain and 77 amino acids in the pre-S1-domain appears to be important for cellular attachmentlations 54. A trlerated 54,56. Myrcludex B is a promising new drug for both hepatitis B and D. That new treatment is a much urgent need in CHD does not need further explanation. The following issues remain to be resolved: (i) optimal dose and duration for use in human CHD needs to be established; (ii) superiority of combination treatment of Myrcludex B with peg-IFN over peg-IFN monotherapy needs to be convincingly shown; (iv) safety appears to be of less concern, but the effect on bile acids has to be explored in a larger patient group; (v) efforts for replacing a daily subcutaneous formulation with an oral formulation should be seriously considered – this would enable long-term use.57, and preclinical studies revealed that they abolished HDV-like particle productionin vitro58 andin vivo59. In the proof-of-concept study, the farnesyltransferase inhibitor lonafarnib (LNF) was tested for a duration of 28 days in 14 CHD patients. A dose-dependent reduction of HDV RNA was observed60. LNF serum concentrations correlated with HDV RNA change. Some characteristics of LNF are provided in61. Boosting with RTN increased serum concentrations of LNF 4- to 5-fold. The combinations of LNF–RTN and LNF–peg-IFN led to serum HDV RNA declines of more than 3 logs10 IU/mL from baseline after 8 weeks of treatment61. Studies on the optimal combination treatment regimen with LNF and RTN and triple therapy with LNF, RTN, and peg-IFN have been tested for different durations in the LOW-R2 study. In this latter study, the triple combination treatment consisting of 25 mg LNF bid with RTN 100 mg bid and peg-IFN appeared to combine best efficacy with tolerability62. Three out of 5 patients were HDV RNA negative after 24 weeks of treatment. LNF at 25 or 50 mg bid dosing with RTN was as effective as all oral combination treatment. Five out of 14 (36%) patients had HDV RNA levels below the limit of quantification at 24 weeks of treatment. Combination of LNF at doses of 75 mg bid and higher as dual or triple therapy was not tolerated because of side effects and also did not lead to better antiviral efficacy62. In the dose-escalation study in Hannover, patients started with a LNF/RTN dose of 50 mg/100 mg bid and the LNF dose was increased first to 75 mg and then to 100 mg bid at roughly 4-week intervals when patients tolerated the administered doses. Dose was not further increased after reaching LNF/RTN (100 mg/100 mg bid). Escalation to this ultimate dose was possible in 10 out of 15 patients. One patient became HDV RNA negative at the end of treatment and another had HDV RNA levels below the quantification level63. Overall, it appears that off-treatment virologic response is lacking in the majority of patients in these studies. An LNF–RTN once-daily dosing combination regimen for 24 weeks was tested in a double-blind approach at the NIH64. Three regimens were tested: LNF 50/75/100 mg qd with RTN 100 mg qd. After 24 weeks of treatment, 6 out of 21 patients had HDV RNA levels below 250 IU/mL. Interestingly, the low-dose group appeared to have better antiviral efficacy compared to higher doses. ALT normalized in all of these studies with LNF in 47 to 75% of patients. LNF is associated with dose-limiting side effects, mainly related to gastrointestinal toxicity. These side effects comprise anorexia, nausea, diarrhea, and weight loss  discontinued treatment at weeks 20 to 27. On long-term off-treatment, only 1 patient had viral and serologic rebound, whereas the other 2 patients continued to be HBV DNA and HBsAg negative. However, REP-2055 intravenous infusion was associated with fever, chills, and headache. REP 2139-Ca, reported to be more stable, was given once-weekly by intravenous infusion to 12 HBeAg-positive CHB patients. Nine responder patients (decrease in HBsAg and HBV DNA by 2 logs) received add-on peg-IFN or thymosin after week 20. During monotherapy, 9 patients had an HBsAg drop of 2 to 7 logs, with 3 becoming HBsAg negative. Similar declines were observed in serum HBV DNA levels. However, off-treatment viral rebound occurred in 7 out of 9 patients within 3 to 24 months68. REP-2139-Mg and REP-2165-Mg, a 2139 derivative with reportedly improved tissue clearance, is being tested in combination with peg-IFN and tenofovir in 20 HBeAg-negative CHB patients pre-treated with a 6-month course of tenofovir alone. Initial data suggest similar declines in HBsAg levels69. All of these studies have relevance to CHD, since HBsAg appears to be selectively targeted. In the only study in CHD with NAPs, REP-2139-Ca was given once weekly with add-on peg-IFN starting at week 15 for another 15 weeks in 12 CHD patients70. Peg-IFN alone was then continued as monotherapy for another 33 weeks. Nine patients displayed declines of >3 logs in HBsAg levels. Post-treatment results were presented at the EASL meeting this year. Five out of 12 patients continued to be HBsAg negative 1 year off-treatment71. Besides administration route-related side effects such as fever, chills, and peripheral hyperemia, other reported side effects include anorexia, hair loss, dysphagia, and dysgeusia, which were attributed by the investigators as being secondary to heavy metal exposure at the trial site  also appears to be a hurdle for this treatment approach.72. A multi-dose extension study (up to 12 doses once-monthly) of the same compound has been conducted. With multiple dosing, additional decline of HBsAg levels was observed, more so in HBeAg-positive than in HBeAg-negative patients73. It was reported that ARC-520 was well tolerated, but the study was put on hold owing to toxicity problems related to the carrier molecule.An exciting concept is the use of small interfering RNAs (siRNAs). ARC-520, a siRNA designed to reduce all HBV transcripts via RNA interference, dose-dependently decreased HBsAg levels after one single injection in HBeAg-negative CHB patients in a phase IIa clinical trial74, anti-HB immune complexes75, and the use of immunologically active adjuvants such as beta-glucosylceramide76. Toll-like receptors (TLRs) as inducers of type 1 IFN responses have always raised interest. Several preclinical studies confirmed their key role in the induction of the innate immune system77. The oral TLR-7 agonist GS-9620 was well tolerated and led to the induction of peripheral ISG15 production in CHB patients78. No effect on HBV DNA was observed, which may be attributable to short treatment duration.Such approaches may have regained interest in CHB, but a breakthrough clinical success is still awaited. Therapeutic vaccination strategies have not been successful so far. New approaches include DNA vaccines79. Hepatotoxicity and off-target immune activation are concerns. The future of immune therapy may be using combination immune therapy such as a PD1 blocker with DNA immunization and NA treatment. Such an approach may deserve serious consideration80.Drugs targeting programmed death protein 1 (PD1) and its ligand (PD-L1) are receiving attention in both cancer immunotherapy and chronic viral hepatitis. Immune checkpoints are exploited by viral pathogens, as shown by T-cell exhaustion in these conditions. Blocking PD1 appears to be a rational goal to activate the HBV-specific T-cell response. In a phase Ib clinical study, the immune checkpoint inhibitor nivolumab was well tolerated and was associated with a significant decline in HBsAg levels after a single dose of nivolumabsiRNAs and various immunological treatment approaches represent scientifically sound treatment vehicles. They are based on a solid scientific background. We know, for example, from hepatitis B that off-treatment viral clearance necessitates tight immune control, hence an immune-based approach in controlling CHD makes sense. However, data showing viral efficacy are needed. The lack of success so far points to the complexity of the immunological process. Maybe better surrogate markers of immunological activity are needed beforehand. Similarly, siRNAs deserve attention, as with one formulation several siRNAs targeting different pre-genomic RNAs can be used. However, for both the immunological and the oligonucleotide approach, time is needed for further development of these approaches.CHD represents the only hepatotropic viral infection where new drugs have not entered clinical practice despite being the most severe form of chronic viral hepatitis. Current treatment with IFNs is unsatisfactory. Hence, new treatment strategies are urgently required. Hepatocyte entry inhibitors, prenylation inhibitors, and NAPs represent a glimmer of hope for patients suffering from CHD. It may be said that none of the new approaches are comparable in efficacy and safety to new drug treatments such as entecavir or tenofovir in hepatitis B and several very effective direct-acting antivirals in hepatitis C. However, their first-generation counterparts also had several deficiencies. From the practicing clinician’s and patient’s point of view, despite their shortcomings, these new treatment approaches for CHD have to be developed fast, since at the current stage treatment with IFNs is effective in only a small proportion of patients, and the situation is further complicated by the decreased motivation and willingness of physicians to consider prolonged courses of peg-IFN treatment and as such some patients who would have benefited from peg-IFN treatment lose their chance. It is difficult to say which of the approaches is going to be the best option for the treatment of CHD, but it is likely that one, two, or all of the drugs reported above will be used in combination with peg-IFN. As such, CHD will probably remain the one viral hepatitis infection where IFN treatment will continue to be a part of its management.


Purpose: To determine if topical beta-blocker use is associated with increased cardiovascular mortality, particularly among people with self-reported glaucoma.Methods: All participants who participated in the first health check  of the European Prospective Investigation into Cancer (EPIC) Norfolk cohort (1993–2013) were included in this prospective cohort study, with a median follow-up of 17.0 years. We determined use of topical beta-blockers at baseline through a self-reported questionnaire and prescription check at the first clinical visit. Cardiovascular mortality was ascertained through data linkage with the Office for National Statistics mortality database. Hazard ratios (HRs) were estimated using multivariable Cox regression models. Meta-analysis of the present study’s results together with other identified literature was performed using a random effects model.Results: We did not find an association between the use of topical beta-blockers and cardiovascular mortality . In the 514 participants with self-reported glaucoma, no association was found between the use of topical beta-blockers and cardiovascular mortality . In the primary meta-analysis of four publications, there was no evidence of an association between the use of topical beta-blockers and cardiovascular mortality .Conclusion: Topical beta-blockers do not appear to be associated with excess cardiovascular mortality. This evidence does not indicate that a change in current practice is warranted, although clinicians should continue to assess individual patients and their cardiovascular risk prior to commencing topical beta-blockers. Additional cardiovascular mechanisms include dysrhythmias and impaired cardiac output,8 although investigations into the association between topical beta-blocker use and cardiovascular mortality show conflicting results. The Blue Mountains Eye Study2 reported an increase in cardiovascular mortality in glaucoma patients using topical beta-blockers, whereas the Rotterdam Study9 found non-significant associations with long-term exposure. Given the significant global burden of cardiovascular disease, the widespread use of topical beta-blockers10 as a cost-effective treatment, and the availability of off-patent effective alternatives in the form of prostaglandin analogues,11 there is clear justification to explore the question further.Topical beta-blockers remain one of the primary treatments for glaucoma.This study aimed to investigate the effects of topical beta-blockers on cardiovascular mortality. First, we examined the association in the European Prospective Investigation into Cancer (EPIC) Norfolk Cohort Study. Second, we conducted a systematic review of the literature and pooled our results with previously published data in a meta-analysis.12 In brief, all persons aged 40–79 years in databases of the 35 participating general practices in the region were sent invitations . Consent was received from 30,445 participants, of which 25,639 attended the first health examination.12 The Norfolk Local Research Ethics Committee approved the EPIC-Norfolk study, and all volunteers gave written informed consent.EPIC-Norfolk is a large, prospective, cohort study. All participants were invited to attend via their general practitioner (GP) between March 1993 and December 1997. The participation rate was 33%. As nearly all residents in the United Kingdom are registered with a GP through the National Health Service, general practice lists serve as population registers. Detailed methods have been published elsewhere.12 All EPIC-Norfolk participants were followed up for mortality and flagged for death certification at the Office for National Statistics, with vital status ascertained on the whole cohort up until 30 June 2013.12 Coding of death certificates was performed by trained nosologists according to the International Classification of Diseases (ICD). Cardiovascular mortality was defined as any individual with ICD codes 410–448 (ICD 9th revision) or ICD codes I10–I79 (ICD 10th revision) listed as cause of death.We ascertained topical beta-blocker use through completion of a detailed health and lifestyle questionnaire and by asking participants to bring all previous prescriptions and medications they were taking at baseline to the health examination, where prescriptions and medicines were checked by trained nurses.12 The following covariables were measured through a self-reported questionnaire: History of angina, cholesterol, diabetes, education, glaucoma, myocardial infarction, stroke, hypertension, smoking and social class. Glaucoma ascertainment was also self-reported through direct questioning on the questionnaire. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were recorded during a health examination as the mean of two measurements taken from the right arm with the participant seated for 5 minutes, using an Accutorr Plus blood pressure monitor .The design and characteristics of EPIC-Norfolk assessment methods for all covariables of interest have been described previously.a priori assumptions of known confounders in the final Cox model. Raised cholesterol was considered to be a potential mediator on the causal pathway5–7 and therefore not included in the final model. A subgroup analysis was performed to look at the association between topical beta-blocker exposure and cardiovascular mortality in only participants with self-reported glaucoma. Multiple sensitivity analyses were carried out to explore the robustness of our findings. The effect of different covariables, glaucoma status, cholesterol as a confounder rather than a mediator, whether having manifest cardiovascular disease might alter exposure patterns and introduce reverse causation were explored. No adjustments were made for change in exposure status over the course of the study period. This allowed direct comparison with methods from the Blue Mountains Eye Study2 and Rotterdam Study9. However, as glaucoma is an age-related disease,13 and given the older age range within the EPIC-Norfolk cohort,12 it is probable that further incident cases of glaucoma and thus topical beta-blocker exposure occurred. Therefore, we used census-derived population data, together with epidemiological models to estimate the potential impact of incident glaucoma during the follow-up period. We also examined the association between topical beta-blocker use and cardiovascular mortality at the second health check (1998–2000) to explore the effect of changing treatment during the follow-up period.Differences in characteristics of participants by exposure status were evaluated using Chi-squared and Welch’s unpaired T-tests. All participants with missing covariable data were included in the initial analyses, with a complete case analysis conducted on the final models. Associations between use of topical beta-blockers and cardiovascular mortality were assessed using Cox proportional hazard models. All analyses were adjusted for age and sex. Potential confounders were selected using univariable comparisons of covariables, and variables which were significantly associated with both exposure and outcome were included. We also included covariables based on A literature-based meta-analysis was performed to look for published evidence on the association between topical beta-blockers and cardiovascular mortality. The search strategy was performed within PubMed .The following terms were used in the literature search strategy in July 2014;(1) topical[All Fields], (2) “adrenergic beta-antagonists” [Pharmacological Action] OR “adrenergic beta-antagonists”[MeSH Terms] OR (“adrenergic” [All Fields] AND “beta-antagonists”[All Fields]) OR “adrenergic beta-antagonists” [All Fields] OR (“beta” [All Fields] AND “blockers” [All Fields]) OR “beta blockers”[All Fields], (3) “glaucoma”[MeSH Terms] OR “glaucoma”[All Fields], (4) “mortality”[Subheading] OR “mortality”[All Fields] OR “mortality”[MeSH Terms], (5) (“cardiovascular system”[MeSH Terms] OR (“cardiovascular”[All Fields] AND “system”[All Fields]) OR “cardiovascular system”[All Fields] OR “cardiovascular”[All Fields]) AND , (6), (1) AND (2), (7) (3) OR (6), (8) (4) OR (5). The search (7) AND (8) returned 278 publications.in-vitro experiments, ecological studies, case series, case reports, opinion pieces, letters, did not look at topical beta-blockers as an exposure and did not look at cardiovascular mortality as an outcome. Publications were additionally excluded if they were reviews of published associations with no new contributing data.Publications were reviewed if they reported associations between topical beta-blocker use and cardiovascular mortality. No exclusion criteria were applied to language or journal publication date. Publications were excluded if they were non-human studies, The exclusion process is detailed in A random effects model was used to pool the results and accommodate between-study heterogeneity. Between-study heterogeneity was analyzed using the I-squared test statistic. All analyses were carried out in Stata 12.1 .A total of 25,639 participants with a mean age of 59.2 years  were included in this analysis. Participants were followed up for a median of 17.0 years and a total of 407,524 person-years. During the follow-up period, 6525 individuals died (25.4%) of which 2158 deaths (33.1%) were attributed to cardiovascular disease. A total of 19,114 individuals were alive at the time of follow-up. Topical beta-blockers were used by 179 individuals (0.7%) of whom 82 (45.8%) had died during follow-up. Of these 36 (20.1%) participants who used topical beta-blockers died of cardiovascular causes.14 numbers with incident glaucoma were calculated from this cohort and applied to the aging EPIC-Norfolk cohort. Non-conservative models predicted a 1.56% incident rate of glaucoma. Applied to the baseline population excluding those with self-reported glaucoma, this predicted an additional 392 cases of incident glaucoma over the study period. Trends in glaucoma medication prescribing have changed with time, where prostaglandins are the preferred medication15 and beta-blocker prescription has dropped by a factor of four. Applying an exaggerated 25% prevalence of topical beta-blocker exposure to these incident cases15 would lead to an additional 98 topical beta-blocker exposures in the EPIC-Norfolk cohort. Applying an exaggerated death rate of 50% to those newly diagnosed with glaucoma on topical beta-blockers, would add an additional 49 deaths. Assuming 30% died from cardiovascular causes compared to the 20.1% observed in the present study, along with adjustments in exposure status in baseline users through application of historic trends in prescribing,15 did not yield significant associations of cardiovascular mortality . This would suggest the findings of this study are robust to changes in exposure status over time.At baseline, 514 participants (2.0%) had glaucoma of which 229 (44.6%) died during follow-up. Of these 92 participants (17.9%) died of cardiovascular causes. Overall, 285 participants with glaucoma were alive at the end of the study. 2,3,9 that looked at topical beta-blockers and cardiovascular mortality were identified from the systematic literature search. 2 statistic was 1.1% (p = 0.387). Topical beta-blocker use was not associated with cardiovascular mortality .A total of three studies,In this prospective cohort study of 25,639 participants, topical beta-blocker use was not associated with increased cardiovascular mortality. Furthermore, in the 514 participants within the cohort who had self-reported glaucoma, we did not detect an association between the use of topical beta-blockers and a higher risk of cardiovascular mortality. There was also no evidence of an association between the use of topical beta-blockers and cardiovascular mortality. These results do not support higher cardiovascular mortality from topical beta-blocker use. However, the current study is based on self-reported glaucoma, which is a key limitation.9 but differ from the Blue Mountains Eye Study6 and Barbados Eye Studies.3 In the Blue Mountains Eye Study, the association between topical beta-blockers and cardiovascular mortality was only seen in participants using timolol with glaucoma, compared to those not using topical timolol without glaucoma. The effect on cardiovascular mortality was not shown in baseline users of timolol before stratification by glaucoma status . In baseline timolol users in the Barbados Eye Studies, there was also no association shown between timolol use and cardiovascular mortality . The association was only seen when compared within users diagnosed with open-angle glaucoma. The primary meta-analysis used these summary statistics in order to compare like with like and did not show an association between topical beta-blocker use and cardiovascular mortality. Subgroup analysis of participants with self-reported glaucoma in the present study found no association between exposure to topical beta-blockers and cardiovascular mortality .Results from this EPIC-Norfolk study are in agreement with the Rotterdam Study16–20 However, the majority of these studies have shown the protective effect only in participants who are post myocardial infarction or with established cardiovascular disease. These observed benefits may not translate to people with no pre-existing cardiovascular disease. The main cardiovascular effects of beta-blockers include reduction of heart rate, myocardial contractile force, cardiac output and peripheral vasoconstriction. Unwanted side effects include bradycardia and exacerbation of heart failure in susceptible patients, and these complications form a potential mechanism for increased risk of cardiovascular mortality from topical beta-blocker use, although there is little observed evidence of this beyond case reports.8 Randomized controlled trials have shown that topical beta-blockers may have an adverse effect on lipid profiles. A review of timolol, the most common topical beta-blocker linked to adverse lipid profiles, found that when given systemically, timolol reduced mortality after myocardial infarction21 indicating that any deleterious effects on serum lipids did not attenuate its protective effect on the heart.There is well-established literature on the protective effects of oral beta-blockers. Multiple randomized controlled trials and systematic reviews have found that use of systemic beta-blockers decreases mortality, improves survival, and is cardio-protective.22 has confirmed accuracy of cardiovascular mortality in EPIC-Norfolk, with 38 of 39 cases confirmed as correct. Last, the linking of outcome data through the Office for National Statistics death register allowed for complete ascertainment of outcome status in this study, regardless of whether the participant was lost to follow-up. This eliminated attrition bias.The strengths of this study lie in its study design; EPIC-Norfolk is a long-term prospective cohort study. Other strengths include the long period of follow-up, the number of participants, and the high number of incident cardiovascular mortality events, enabling high statistical power. The large number of covariables measured at baseline allowed for adjustment of multiple confounders in the analysis and the assessment of the robustness of the findings across different analytical variations. A validation studyn = 514). This was further reduced after excluding deaths not from cardiovascular causes (n = 377) and may have allowed type II error to occur. There may be a degree of healthy-volunteer bias occurring. This is suggested by the considerably lower proportion of current and ex-smokers, and a higher proportion of never smokers compared to the rest of England. This may also have contributed to the relatively low proportions of cardiovascular deaths in this older population. The study only assigned participants to the exposed category if topical beta-blockers were used before or at baseline and no adjustments were made for changes in exposure status with time. Additionally, it was not possible to ascertain compliance of individuals exposed to topical beta-blockers, who may have filled prescriptions they were not taking. This may have resulted in an underestimate of effect. Our methods are in line with the previous studies examined in the meta-analysis, and allowed for direct comparisons. We did, however, examine the impact of incident glaucoma and did not detect a significant effect. Publication bias was possible in the meta-analyses, but the use of a funnel plot was of little value due to the number of studies identified. It is plausible that there are smaller studies that have unpublished non-significant results that would strengthen the absence of an association between topical beta-blocker use and cardiovascular mortality and favor the null hypothesis.There were several key limitations of the study. There was limited power within the subgroup analysis of glaucoma users whose baseline cohort was small (23–26 This is particularly relevant to this study as we used self-reported glaucoma, where participants could report a glaucoma diagnosis even if they had ocular hypertension. Furthermore, up to 50% of glaucoma in the community can be undiagnosed.27,28 These considerations would lead to a measurement error in exposure ascertainment, and although likely to contribute to a null finding, the exact impact is unknown.We did not examine the relationship between glaucoma diagnosis and mortality as it was beyond the scope of this study. It is possible that topical beta-blocker use may mask heterogeneous pathophysiological processes that underpin both glaucoma and ocular hypertension. Both conditions are treated with topical beta-blockers and display different associations with hypertension and diabetes.In conclusion, no association was observed between topical beta-blocker use and cardiovascular mortality in all participants or in participants with self-reported glaucoma. A meta-analysis with other large population-based studies showed no association in the pooled estimates between topical beta-blockers and cardiovascular mortality, both for all participants and for only participants with glaucoma. However, further studies are required that allow for adjustment for changes in exposure to topical beta-blockers. We have found no further evidence to suggest that patients using topical beta-blockers experience excess cardiovascular mortality, and no indications that current clinical practice requires revision. Clinicians should continue to exercise caution when assessing individual glaucoma patients and their cardiovascular risk prior to commencing topical beta-blockers.


Major depressive disorder (MDD) is approximately twice as common in females than males. Furthermore, female patients with MDD tend to manifest comorbid anxiety. Few studies have explored the potential anatomical and functional brain changes associated with MDD in females. Therefore, the purpose of the present study was to investigate the anatomical and functional changes underlying MDD in females, especially within the context of comorbid anxiety.N = 35) and healthy female controls . The severity of depression and anxiety were evaluated by the Hamilton Depression Rating Scale (HAM-D) and the Hamilton Anxiety Rating Scale (HAM-A), respectively. Structural and resting-state functional images were acquired on a Siemens 3.0 Tesla scanner. We compared the structural volumetric differences between patients and HC with voxel-based morphometry (VBM) analyses. Seed-based voxel-wise correlative analyses were used to identify abnormal functional connectivity. Regions with structural deficits showed a significant correlation between gray matter (GM) volume and clinical variables that were selected as seeds. Furthermore, voxel-wise functional connectivity analyses were applied to identify the abnormal connectivity relevant to seed in the MDD group.In this study, we recruited antidepressant-free females with MDD (Decreased GM volume in patients was observed in the insula, putamen, amygdala, lingual gyrus, and cerebellum. The right amygdala was selected as a seed to perform connectivity analyses, since its GM volume exhibited a significant correlation with the clinical anxiety scores. We detected regions with disrupted connectivity relevant to seed primarily within the cortico-striatal-pallidal-thalamic circuit.Amygdaloid atrophy, as well as decreased functional connectivity between the amygdala and the cortico-striatal-pallidal-thalamic circuit, appears to play a role in female MDD, especially in relation to comorbid anxiety. Epidemiological studies have consistently demonstrated gender differences in prevalence rates of unipolar major depressive disorder (MDD), with females being twice as likely than males to develop it , 2. The Recently, researchers have investigated structural and functional neurological differences between female patients with MDD and healthy controls (HC) to better understand the neurobiological mechanisms of MDD , 5. For More recently, resting-state functional connectivity analyses have been used to capture the interactive level of neural systems when no task is being performed , 17. SeeTo identify brain abnormalities of depression in a systemic manner, we combined the voxel-based morphometry (VBM) and resting-state functional connectivity approach to investigate structural alterations and potentially disrupted functional connectivity, respectively. According to evidence from previous neuroimaging studies, we hypothesized that the structural alterations and disrupted connectivity of depression would be mainly observed in the cortico-striatal-pallidal-thalamic circuit. Additionally, given that female patients with MDD tends to show increased anxiety, including higher rates of comorbid anxiety disorders , we furtAll study procedures were approved by the medical Ethics committee of ZhongDa Hospital, Southeast University. Prior to obtaining consent, the capacity to provide informed consent for all potential participants was ascertained by two licensed psychiatrists. After explaining the study procedures, informed written consent was obtained from all participants. All study procedures were conducted in strict accordance with the Declaration of Helsinki.A total of 35 antidepressant-free females with MDD (aged 44.54 ± 11.14 years) from the Department of Psychosomatics and Psychiatry, ZhongDa Hospital, Southeast University and 23 matched HC (aged 39.09 ± 14.35 years) were recruited between March 2012 and December 2014 via community advertisements. Participants were carefully screened in a semi-structured interview by two trained senior psychiatrists.Participants in the MDD group were confirmed to meet the DSM-IV criteria for MDD . The diaHC were physically healthy, had no history of severe illness, and no first degree relative with a history of psychiatric illness. Clinical assessments for the control group were conducted by an experienced psychiatrist in order to verify inclusion and exclusion criteria. Eligible participants were required to have a HAM-D score less than 8 points and to have met the exclusion criteria (1) and (3). Further, all participants were right-handed and were required to have no contraindications for MRI studies.2). Whole brain resting-state fMRI data were acquired using a gradient-recalled echo-planar imaging pulse sequence . During the 8-mintue resting-state fMRI scan, participants were instructed to lay still in the scanner, keep their eyes open, and refrain from falling asleep.Imaging data were acquired on a Siemens 3.0 Tesla scanner with a 12-channel head coil. High-resolution 3-dimensional T1-weighted 3D scans were recorded in a magnetization prepared rapid gradient echo (MPRAGE) sequence  were smoothed with an 8-mm full-width at half-maximum Gaussian kernel.VBM analyses were carried out using Statistical Parametric Mapping software  run on Matlab 2014a . First, MR images were segmented into gray matter (GM), white matter (WM), and cerebrospinal fluid (CSF) using the standard unified segmentation module in SPM8 . Second,We compared regional GM volume across the two groups  with a two sample t-test after controlling for age. Statistical parametric maps were generated after multiple comparison analyses . Female patients with MDD were prone to increased anxiety, including higher rates of comorbid anxiety disorders. Consequently, we conducted Pearson correlations between (1) the GM volume of regions with structural deficits, and (2) symptoms of anxiety as measured by the HAM-A summary score to identify the relationship between structural abnormalities and clinical symptoms in female with MDD.3, and spatial smoothing with a Gaussian kernel of 8 mm3 full-width at half maximum. White matter, cerebrospinal fluid (CSF), and motion-relevant parameters were taken as confounds, following the component-based noise-correction method to minimize non-neural influences on functional MRI (fMRI) signals. The whole brain BOLD signal was excluded as a regressor to eliminate erroneous anti-correlations [Connectivity analyses were performed using SPM8 software and the CONN-fMRI Functional toolbox . Brieflyelations . The reselations . Then, Pp < 0.01 and cluster size >251). Similar to our VBM analysis, the mean Z-values of functional connectivity were retrieved from regions with abnormal functional connectivity and were used to compute Pearson correlations with anxiety symptoms as measured by the HAM-A summary score in the females with MDD group.Two-sample t-tests were conducted to compare the functional connectivity between the two groups , controlling for age. Statistical parametric maps were generated after multiple comparison analysis .As female patients with MDD tended to display elevated anxiety traits, we conducted Pearson correlations between anxiety scores and the GM volume of regions showing structural deficits. As In following-up analysis on resting-state brain activity, the right amygdala was chosen as the seed region. Low frequency functional connectivity maps for the whole brain were generated for each group. As p<0.032) and the left pallidus  correlated significantly with anxiety scores . It has been shown that, compared to HC, patients with depression tend to be attentionally-biased toward negatively-valenced stimuli and are less able to process positive information , 41. GivAberrant connectivity between the insula and the amygdala in patients with MDD has also been reported in neuroimaging studies that use seed-based or ICA methods , 42. TheIt is also worth noting that we found decreased connectivity between striatal systems and the amygdala in females with MDD, compared with HC, which is consistent with previous studies on depression . Animal The thalamus, not just a relay station, but also a highly interactive structure that provides selection and transformation to different inputs, has recently been subjected to intense scrutiny in depression , 30, 45.Taken together, our findings shed light on neural mechanisms underlying the higher incidence rates of MDD in females as compared to males. The present study, benefiting from its unique focus on a female population, has outlined a set of brain regions with alterations at both anatomical and functional levels in females with MDD. It has been long suspected that certain brain abnormalities are responsible for the female predisposition for MDD. Hastings et al have demonstrated that females with MDD relative to males with MDD, displayed decreased GM volume in the amygdala . MeanwhiThere are several limitations to our study that should be noted. First, the small sample size may have limited our ability to detect statistical group differences. For example, we did not detect the disrupted connectivity of the ACC relevant to seed, which has consistently appeared in other functional connectivity studies. Second, some potential confounding factors, such as physiological noise (respiratory and cardiac rhythms) and the arousal level of participants during resting state scans were not controlled for in our analyses. Third, we used the AlphaSim correction for multiple comparisons, although options  are also available and could be used. Fourth, our conclusions were based on an all-female cohort. Any attempt to generalize our findings to a male population should be made with caution.Our study applied VBM and resting-state functional connectivity analyses to identify the brain abnormities of females with MDD at a systemic level. The study suggests that amygdaloid atrophy, as well as decreased functional connectivity between the amygdala and the cortico-striatal-pallidal-thalamic circuit, play an important role in female MDD, especially in relation to comorbid anxiety. Our study has added new insight into the female-specific pathological mechanism of MDD, and has emphasized the role of the amygdala in the pathophysiology of MDD in females.S1 Dataset(ZIP)Click here for additional data file.S2 Dataset(ZIP)Click here for additional data file.S3 Dataset(ZIP)Click here for additional data file.S4 Dataset(ZIP)Click here for additional data file.S5 Dataset(ZIP)Click here for additional data file.S6 Dataset(ZIP)Click here for additional data file.S7 Dataset(ZIP)Click here for additional data file.


Growth failure is common among HIV-infected infants, but there are limited data on the effects of HIV exposure or timing of HIV acquisition on growth.Fourteen thousand one hundred ten infants were enrolled in the Zimbabwe Vitamin A for Mothers and Babies trial in Zimbabwe before the availability of antiretroviral therapy or co-trimoxazole. Anthropometric measurements were taken from birth through 12–24 months of age. Growth outcomes were compared between 5 groups of children: HIV-infected in utero (IU), intrapartum (IP) or postnatally (PN); HIV-exposed uninfected (HEU); and HIV unexposed.Growth failure was common across all groups of children. Compared with HIV-unexposed children, IU-, IP- and PN-infected children had significantly lower length-for-age and weight-for-length Z scores throughout the first 2 years of life. At 12 months, odds ratios for stunting were higher in IU  and IP infants  than in PN infants . Compared with HIV-unexposed infants, HEU infants at 12 months had odds ratios for stunting of 1.23 (95% CI: 1.08–1.39) and wasting of 1.56 (95% CI: 1.22–2.00).HIV-infected infants had very high rates of growth failure during the first 2 years of life, particularly if IU or IP infected, highlighting the importance of early infant diagnosis and antiretroviral therapy. HEU infants had poorer growth than HIV-unexposed infants in the first 12 months of life. Morbidity and mortality are associated with timing of HIV infection, with infants infected IU or IP having poorer outcomes than those infected PN.6 Infants who are exposed to maternal HIV but remain uninfected [HIV-exposed uninfected (HEU) infants] are also at risk of poorer health outcomes than infants not exposed to HIV.7In 2013, an estimated 1.4 million pregnant women were living with HIV worldwide, the majority in sub-Saharan Africa.8 The effect of HIV exposure on growth remains uncertain, with heterogeneous results reported across sub-Saharan Africa.7 The Zimbabwe Vitamin A for Mothers and Babies (ZVITAMBO) trial, which was conducted in Zimbabwe before the availability of ART,9 provides an opportunity to evaluate the growth of infants born to HIV-infected mothers without the confounding effects of ART exposure. We describe linear and ponderal growth of children born to HIV-infected, compared with HIV-uninfected, mothers in Zimbabwe and explore the factors associated with growth failure among HIV-infected  and uninfected (HEU and unexposed) children.HIV-infected infants are at increased risk of low birth weight (LBW) and postnatal growth failure compared with HIV-uninfected infants.12 Briefly, 14,110 mother–infant pairs were enrolled within 96 hours of delivery from clinics in Harare, Zimbabwe. Mother–infant pairs were eligible if the infant was a singleton with birth weight ≥1500 g, the mother planned to stay in Harare after delivery, and neither mother nor infant had an acutely life-threatening condition. Medical care and counseling were offered throughout the trial.13 The trial preceded availability of ART for PMTCT or treatment in Zimbabwe, or recommendations for co-trimoxazole prophylaxis for HIV-exposed infants.The ZVITAMBO trial of peripartum maternal and neonatal vitamin A supplementation was conducted between 1997 and 2001, as reported elsewhere.9 Mothers testing HIV positive at baseline were retested to confirm infection; those who were HIV negative were retested at subsequent blood draws to detect seroconversion. CD4 counts were measured for HIV-positive mothers and a random selection of HIV-negative mothers at baseline using FACSCount . Hemoglobin was measured at baseline using a hemoglobinometer  in all mothers enrolled from October 1998. For all children born to HIV-positive mothers, cell pellets and plasma were prepared from whole blood collected at baseline and all follow-up visits and stored at −70°C. At the end of the follow-up period, the last available sample from each child was tested for HIV by GeneScreen  enzyme-linked immunosorbent assay on plasma if ≥18 months of age or by DNA polymerase chain reaction (PCR) assay Roche Amplicor version 1.5  in cell pellets if <18 months of age. If the last available sample was negative for HIV, the child was classified as HIV negative; if the last available sample was positive, earlier samples were tested to ascertain timing of infection. Children were classified into one of the 5 HIV-exposure categories: (1) unexposed: mother tested HIV negative at baseline; (2) HEU: mother HIV positive at baseline, infant HIV negative; (3) IU infected: infant PCR positive at baseline; (4) IP infected: infant PCR negative at baseline and PCR positive at 6 weeks of age and (5) PN infected: infant PCR negative at 6 weeks and HIV positive at a subsequent time point. Infants in the IP and PN groups contributed data to the HEU group before HIV acquisition. Infants were censored at their last HIV test result to avoid potentially misclassifying infants who had become infected before death or before the end of the study.Mothers were tested for HIV by enzyme-linked immunosorbent assay and western blot using previously described methods.14 Gestational age was calculated using the Capurro method.15 Length and weight were measured at follow-up visits conducted at 6 weeks, 3 months and every 3 months to 12–24 months. Length and weight measurements for each child were converted to length-for-age (LAZ) and weight-for-length Z scores (WLZ) using World Health Organization international standards.15 Based on detailed feeding questionnaires, infants were classified into one of the 4 groups: nonbreastfed, exclusively breastfed , predominantly breastfed (breast milk and nonmilk liquids) and mixed breastfed (breast milk plus nonhuman milks and/or solid foods), as previously described.10 This classification was limited to the first 3 months of life, because by 6 months, 93% of infants were mixed breastfed.10At baseline, infant birth weight was measured using an electronic scale ; infant birth length and maternal mid-upper arm circumference (MUAC) were measured according to methods described by Gibson.The growth patterns of children from birth to 24 months were described by plotting mean LAZ and WLZ against age in months for each HIV infection/exposure group. Univariate logistic regression models were used to evaluate odds ratios (ORs) for stunting and wasting at baseline, 6 weeks, and 6, 12, 18 and 24 months, with HIV-unexposed children as the reference group.For HIV-exposed and PN-infected children, time-varying exposure categories were used at each time point . PN infants were only defined as PN infected at the first positive HIV test. As described above, infants were censored from analyses at the last HIV test result.P < 0.05 were retained in the models for each HIV-exposure group. All statistical analyses were conducted using STATA version 11.1 .To identify factors associated with growth failure, a multivariable generalized estimating equation linear regression model for LAZ was constructed for each HIV infection/exposure group using an exchangeable correlation structure, taking into account within-child correlation between repeated measurements to create growth profiles between 6 weeks and 24 months. For the purposes of these models, infants were categorized based on infection/exposure status at 12 months of age (time-varying status not used). The covariates included in the models were independent predictors in univariate analyses that were not directly related to the definition of the child growth outcomes: infant sex, gestational age and feeding pattern at 3 months of age; maternal CD4 count, hemoglobin, parity , MUAC, death, marital status and education; household income; and vitamin A treatment arm. Only variables that were significant at Mothers provided written informed consent to take part in the trial, as approved by the Medical Research Council of Zimbabwe, Medicines Control Authority of Zimbabwe, Johns Hopkins Bloomberg School of Public Health Committee on Human Research and Montreal General Hospital Ethics Committee.At baseline, 4495 mothers were HIV positive and 9561 were HIV negative. Fifty-one had indeterminate results, and 3 were not tested; infants of these mothers were excluded. Of the 9561 mothers who were HIV negative at baseline, 351 seroconverted during follow-up; infants of these mothers were excluded. Thirty-three infants born to HIV-positive mothers were never PCR tested; these infants were excluded. Taken together, 438 (3.1%) of the 14,110 ZVITAMBO infants were removed from this current analysis, leaving 13,672 infants who were classified based on maternal HIV status and final infant HIV status as IU, IP, PN, HEU and unexposed. The number of participants in each group varied throughout the study period because (1) a proportion of HEU infants became HIV infected (IP or PN); (2) infants that acquired HIV with uncertain timing of infection were included in the analysis only at time points where their HIV status was certain; (3) study visit completeness was not 100%; (4) loss to follow-up and infant deaths occurred and (5) infants with incomplete results were censored at their final HIV test. Number of participants (N) included at each time point are shown in tables and figure legends.Table At birth, HIV-infected and HEU infants had significantly lower LAZ and WLZ and had more stunting and wasting than HIV-unexposed infants Tables  and 3. CThere was a substantial drop in LAZ between birth and 6 weeks in all groups and a steady decline thereafter to around 21 months of age, with the drop being most pronounced in HIV-infected infants  was common among all children in the study, but was especially frequent among HIV-infected children  was uncommon among children born to HIV-uninfected mothers, but was more prevalent in HEU infants, and was especially frequent among HIV-infected children Table . HIV-infTo understand the mechanisms underlying growth failure, we undertook generalized estimating equation models for each exposure category, identifying factors associated with linear growth profiles between 6 weeks and 24 months , found a 1.85-fold increased relative risk (RR) of stunting among HEU infants in the first year of life (95% CI: 1.03–3.31). Conversely, HIV-unexposed children had more stunting than HEU children between 1 and 2 years of age, which coincided with the weaning of HIV-unexposed children ; after 2 years of age, HEU children were again at increased risk of stunting .41 Children were not followed past 2 years of age in the ZVITAMBO trial, but notably, breastfeeding status was not associated with linear growth of HEU infants.A key finding from this study is that HEU infants had poorer growth than HIV-unexposed infants during infancy. Consistent with most42 It is important to note that this study was conducted in the pre-ART era and infants were therefore exposed to untreated maternal HIV. However, we found no association between maternal disease severity (measured as CD4 count) and HEU infant growth, in contrast to mortality outcomes for this cohort.4 In Kenyan HEU children studied before ART availability, there was a 30% increased risk of stunting per log increase in maternal viral load,43 and in South African HIV-exposed infants, stunting, underweight and wasting were 1.7-fold, 1.6-fold and 1.6-fold higher, respectively, in infants born to mothers with CD4 <200 cells/μL compared with mothers with higher CD4 counts, suggesting that severity of maternal HIV can influence child growth.The mechanisms underlying growth faltering among HEU infants in this cohort are unclear. Although it could be argued that stunting in HEU infants is more likely related to adverse social conditions rather than to HIV-related biologic mechanisms, maternal death, income, education and parity were not related to linear growth of HEU infants in this study. The relative contributions of factors associated with HEU infant growth remain uncertain. As mean LAZ in HEU infants was already lower at birth, the fetal environment may have a key influence on infant growth; HEU fetal growth restriction, and therefore infant growth restriction, may be at least partly driven by maternal HIV infection and inflammation during pregnancy.This study has several strengths and limitations. Timing of infant HIV infection was carefully characterized, allowing us to evaluate differences in growth between IU, IP and PN groups. This is the largest cohort of HEU children to date, with a suitable comparison group of HIV-unexposed children. We censored infants at the last available HIV test, to ensure that the HEU group did not include undiagnosed PN children. However, very LBW infants (<1500 g) were excluded from the trial; because LBW was associated with postnatal growth, many infants excluded on this basis may have had poor postnatal growth. We used only WLZ to assess wasting in children, not MUAC; this could have influenced the proportion of children categorized as wasted. The high mortality among children may have led to survivor bias, especially among those with early HIV acquisition.In summary, we show that growth failure is highly prevalent in Zimbabwean infants. In the absence of ART, HIV-infected infants had strikingly high rates of stunting and wasting, and earlier HIV infection was associated with poorer growth. Infants born to HIV-infected mothers, even in the absence of vertical HIV transmission, had poorer growth throughout the first year of life compared with those born to HIV-uninfected mothers. This study provides a strong rationale for strengthening efforts to eliminate mother-to-child HIV transmission and improving access to early infant diagnosis and co-trimoxazole prophylaxis, with ART initiation among those who become HIV-infected. These data suggest that early interventions are critical to tackle growth failure in sub-Saharan Africa.


A novel system to cultivate and record from organotypic brain slices directly on high-density microelectrode arrays (HD-MEA) was developed. This system allows for continuous recording of electrical activity of specific individual neurons at high spatial resolution while monitoring at the same time, neuronal network activity. For the first time, the electrical activity patterns of single neurons and the corresponding neuronal network in an organotypic hippocampal slice culture were studied during several consecutive weeks at daily intervals. An unsupervised iterative spike-sorting algorithm, based on PCA and k-means clustering, was developed to assign the activities to the single units. Spike-triggered average extracellular waveforms of an action potential recorded across neighboring electrodes, termed “footprints” of single-units were generated and tracked over weeks. The developed system offers the potential to study chronic impacts of drugs or genetic modifications on individual neurons in slice preparations over extended times. Key ConceptsElectrical-activity map: A heat map that is plotted according to the largest absolute values of the negative amplitudes of extracellular action potentials on each electrode.Footprints: A single-unit footprint represents the spatiotemporal extracellular waveforms of a single neuron, which has been calculated by spike-triggered averaging of signals determined according to spike times at the electrode with the largest signal.(CF amplitude):Central footprint peak amplitude  the absolute value of the largest negative spike amplitude, within the footprint.(CF location):Central footprint location  the location of the largest amplitude-signal within the footprint.(CF area):Central footprint area  the area of footprints that included electrodes with negative spike amplitudes larger than 50% of the CF amplitude.ex vivo system, and bridge the gap between dissociated cell cultures and in vivo animal experiments. Organotypic hippocampal slice cultures partially preserve the cytoarchitecture, synaptic circuits, and chemical signaling of the hippocampus  23. The locations and magnitudes of peak amplitudes in the footprints and the extensions of the central footprint areas were compared between every two consecutive days. The results showed that the single-unit footprints remained relatively stable over days.In the following sections, we will describe a method for recording spontaneous activity of hippocampal slice cultures by means of HD-MEAs at high spatiotemporal resolution over extended cultivation times. The aim was to track network activity as well as neuronal action potentials at single-cell and sub-cellular resolution. To achieve the goal of recording from the same slice culture at multiple time points over weeks, specific slice culture chambers and rotation racks, which can accommodate the HD-MEAs, were designed Figure . ExtraceAll cell material acquisition and animal experiments were done in accordance with guidelines approved by the Basel-Stadt veterinary office according to Swiss federal laws on animal welfare (protocol number: 2358).B6.Cg-Tg (Thy1-YFP) 16Jrs/J, Feng et al., C57 BL/6) cross. Genotyping was done through PCR analysis.Thy1-YFP-16 mice (2 + 5% CO2) HBSS , mixed with D-Glucose , Kynurenic acid , and penicillin-streptomycin  under sterile conditions. Bi-lateral hippocampi were dissected and embedded in low-melting-temperature agarose solution . Sagittal hippocampal slices (300 μm thickness) were obtained by using a vibratome (Leica VT1200 S). The HD-MEA chip was sterilized in 70% ethanol for 40 min and coated with 0.05% PEI . Culture medium was replaced after 3 days with culture medium containing penicillin-streptomycin without B27.Newborn Thy1-YFP mice, aged between postnatal day 5–7, were used to obtain brain slice cultures. The details of the applied organotypic culturing procedure have been described in previous papers . A plastic ring  in a solution containing 7 mM hexachloroplatinic acid, 0.3 mM lead acetate, and hydrochloric acid with an adjustment of the solution pH to 1 -based, planar, high-density microelectrode array (HD-MEA) was designed and produced in our labs . Before starting experiments, the slices were checked for activity , and a threshold detection was applied (5.5 times the noise standard deviation). A MATLAB (R2012a and R2014b) code was used for data analysis .The global network activity of the slice cultures was evaluated with two different methods: (1) by using the “electrical-activity map” without spike sorting; (2) by assessing the total number of neurons that could be detected through spike sorting of the high-density block recordings. To generate the electrical-activity map of the slice culture, the largest absolute values of the negative amplitudes of the extracellular action potentials on each electrode were plotted  for each of the recording electrodes.Negative spike amplitudes were ranked for each electrode. The electrode with the largest spikes was selected as a “core electrode” Figure .As neuronal action potentials could be detected on several neighboring electrodes simultaneously, the spike times detected from the core electrode were used to investigate recordings of neighboring electrodes Figure .From these, the 10 electrodes recording the largest signals were selected to assist spike sorting.k-means  a silhouette value was obtained for each spike waveform to assess how well each spike belongs to its cluster. (ii) The mean of all silhouette values was computed. The cluster with the largest mean of silhouette values was chosen  to II (a) were repeated.Single-unit footprints were established for all recording blocks over the whole array.Step III. Merging templates within and across configurations:To assess whether the obtained single-unit footprints originated from the same neuron, the spike waveforms of all detected single-unit footprints were compared, and a matching coefficient value Figure  was calcThe matching-coefficient method Figure  was alsoThe single-unit footprints tracked on different days were presented as heat maps Figure . Single-Missing parts of a single-unit footprint can be an artifact of the recording procedure, in which not all electrode blocks were recorded at precisely the same time. Missing neuron parts could occur, if the neuron had not been spontaneously active while the respective electrode block (in which the neuron or parts of the neuron were located) was recorded. Then, upon merging the electrode blocks, the respective neuron part would be missing. The consequences of this recording artifact can, for instance, be seen in Figure CF amplitude)central footprint peak amplitude . A threshold of 50% of the largest-amplitude signal   was compared, and calculated as:The distance that the CF location movement analysis. The group mean was used for the CF amplitude analysis and the CF area analysis. In total, data from 22 single-units tracked in 3 different cultures between DIV 16–21 were used to analyze the single-unit footprint dynamics.The group medians were compared across days for the 2 level (5%).To observe the changes in electrical activities of an organotypic slice culture with HD-MEA recordings over extended time periods, a roller-tube setup, adapted to the HD-MEA format, was developed in house. This system allowed for culturing brain slices on HD-MEAs over weeks, while HD-MEA electrophysiology recordings could be performed at any time during the cultivation period. The roller-system consisted of culture chambers and a rotation rack Figure . The culThe results evidenced successful and reliable culturing and recording of organotypic slice cultures on HD-MEAs with the roller-tube method. In total, 32 slice cultures from 7 different preparations were produced. Of these, 26 slice cultures showed spontaneous activity on the first day of HD-MEA recordings. There were 20 cultures (out of the 26 cultures) still showing activity after DIV 10, 12 cultures after DIV 20, and 4 cultures after DIV 30. Five cultures from 2 different preparations were recorded from consistently between DIV 13–23, and these data were used to generate electrical-activity maps and to perform single-unit detection for network analysis. Three of these five cultures were selected for single-unit tracking over days.As expected, the global network activities of the slice cultures decreased while increasing cultivation time. Electrical-activity maps were generated for each slice culture during every recording session, based on the largest absolute signal amplitude (larger than 50 μV) extracted from the spikes on each electrode Figure . The numThe overall network activity of the slice cultures was also characterized in terms of detectable single-units at each recording day. Due to the high electrode density of the HD-MEA, single-unit activities could be identified from the high-density-block recordings with the help of an automatic spike sorting software developed in house. Multiple single-units could be identified within the same high-density recording block, and each single-unit location could be defined according to its nearby electrode positions Figure .The numbCF location, the CF amplitude, and the CF area. The CF locations were compared between every two consecutive recording days . The analysis was performed on 22 neurons, which were tracked between DIV 16–21.In a next step, we wanted to demonstrate that activities of multiple single-units can be identified Figure , separatCF location movement was close to zero  EngCaBra Contract No. 264417, and the European Research Council Advanced Grants 267351 “NeuroCMOS” (FP7) and 694829 “neuroXscales” (Horizon 2020), as well as the Swiss National Science Foundation through Grant 205321_157092/1. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.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.