Despite adequate treatment with antipsychotics, a substantial number of patients with schizophrenia demonstrate only suboptimal clinical outcome. To overcome this challenge, various psychopharmacological combination strategies have been used, including antidepressants added to antipsychotics.To analyze the efficacy of add-on antidepressants for the treatment of negative, positive, cognitive, depressive, and antipsychotic-induced extrapyramidal symptoms in schizophrenia, published randomized controlled trials assessing the efficacy of adjunctive antidepressants in schizophrenia were reviewed using the following parameters: baseline clinical characteristics and number of patients, their on-going antipsychotic treatment, dosage of the add-on antidepressants, duration of the trial, efficacy measures, and outcomes.There were 36 randomized controlled trials reported in 41 journal publications (n=1582). The antidepressants used were the selective serotonin reuptake inhibitors, duloxetine, imipramine, mianserin, mirtazapine, nefazodone, reboxetin, trazodone, and bupropion. Mirtazapine and mianserin showed somewhat consistent efficacy for negative symptoms and both seemed to enhance neurocognition. Trazodone and nefazodone appeared to improve the antipsychotics-induced extrapyramidal symptoms. Imipramine and duloxetine tended to improve depressive symptoms. No clear evidence supporting selective serotonin reuptake inhibitors’ efficacy on any clinical domain of schizophrenia was found. Add-on antidepressants did not worsen psychosis.Despite a substantial number of randomized controlled trials, the overall efficacy of add-on antidepressants in schizophrenia remains uncertain mainly due to methodological issues. Some differences in efficacy on several schizophrenia domains seem, however, to exist and to vary by the antidepressant subgroups—plausibly due to differences in the mechanisms of action. Antidepressants may not worsen the course of psychosis. Better designed, larger, and longer randomized controlled trials are needed. It is well established that antipsychotics are effective in the majority of patients with schizophrenia . HoweverClozapine, the prototypic “atypical” antipsychotic (presently referred to most often as second-generation antipsychotic [SGA]), is proven to be effective in a significant proportion of the patients who do not respond to other antipsychotic medications . The mecIndeed, a number of medications have been studied as adjuncts to antipsychotics with a goal to improve positive, negative, affective, or cognitive symptoms of schizophrenia resistant to antipsychotic medication alone. These pharmacological agents include lithium, anticonvulsants, antiinflammatory and glutamatergic drugs, sex hormones, cholinesterase and phosphodiesterase inhibitors, and various antidepressants .Although the use of antidepressants added to antipsychotics in schizophrenia has been a subject of intensive research during the recent decades, the evidence regarding their efficacy still remains conflicting . NeverthThe present study aimed to review the published randomized controlled trials (RCTs) with antidepressants added to antipsychotics in the treatment of schizophrenia.Published RCTs assessing the efficacy of adjunctive antidepressants in schizophrenia were searched for in the PUBMED, PsycINFO, and PsycLIT databases from January 1960 to December 2013, using the following keywords: “schizophrenia” AND “antidepressant” OR “tricyclic antidepressant” OR “monoaminoxidase inhibitor” OR “selective serotonin reuptake inhibitor” OR “norepinephrine reuptake inhibitor”, as well as “schizophrenia” AND “amitriptyline” OR “imipramine” OR “clomipramine” OR “fluoxetine” OR “fluvoxamine” OR “sertraline” OR “paroxetine” OR “citalopram” OR “escitalopram” OR “venlafaxine” OR “duloxetine” OR “bupropion” OR “milnacipran” OR “reboxetine” OR “trazodone” OR “nefazodon” OR “mianserin” OR “mirtazapine” OR “vortioxetine” OR “vilazodone” OR “agomelatine”, as well as “schizophrenia” AND “double-blind” AND “augmentation”, as well as “schizophrenia” AND “double-blind” AND “adjunctive.” To obtain further data, hand searches of references in published review articles as well as cross-referencing were used.All citations were reviewed using the following parameters: baseline clinical characteristics of patients and their antipsychotic treatment, dose of the add-on antidepressant, duration of the trial, number of participants, efficacy measures, and outcome.We were able to locate a total of 36 RCTs  including 1582 subjects with a diagnosis of schizophrenia or schizoaffective disorder . All incIn all trials, the efficacy measures—Brief Psychiatric Rating Scale, Positive and Negative Syndrome Scale (PANSS), Scale for the Assessment of Negative Symptoms, Scale for the Assessment of Positive Symptoms, Clinical Global Impression Scale, and the Wing’s scale—were used prospectively from baseline to endpoint. In only 14 studies were a priori defined minimum cut-off points on the Brief Psychiatric Rating Scale or PANSS scales used as an inclusion criterion, while in the others the subjects were vaguely defined to be “symptomatic” or “suffering from positive or negative symptoms” despite adequate antipsychotic treatment. Twenty-two trials included clinically stable subjects. Furthermore, in 21 trials, a thoroughly defined period of an unchanged antipsychotic treatment was required prior to participation in the study. This period varied from 1 week to 6 months. Presence of the current significant depressive symptoms served as an exclusion criterion in 12 trials. Of the others, 15 trials used depression rating scales as an outcome measure: the Hamilton Rating Scale for Depression, Calgary Depression Scale for Schizophrenia, Beck Depression Inventory, or Montgomery–Åsberg Depression Rating Scale. Extrapyramidal symptom rating scales—the Simpson-Angus Scale for extrapyramidal effects, the Abnormal Involuntary Movements Scale, and the Barnes Akathisia Scale—were used in 17 trials. Noteworthy, 5 trials were initially designed to investigate the effect of antidepressant add-on treatment of antipsychotic-induced weight gain  or smokiThe add-on antidepressant and add-on placebo groups did not generally differ significantly in baseline clinical characteristics. In only one trial  the add-The included RCTs are presented in P=.07) in favor of an antidepressant was also found in 1of 3 RCTs with mianserin  improved the positive symptoms of schizophrenia . Noteworianserin .No add-on RCTs with novel antidepressants agomelatine, vortioxetine, or vilazodone are yet available.Antidepressant augmentation was found to be effective in the treatment of depressive symptoms of schizophrenia in only a small number of studies. These included imipramine , fluoxetAll trials with the SSRIs  demonstrOf the SSRIs, the only available study  did not reveal any improvement in cognitive functions . Of the The effects of different groups of add-on antidepressants on major psychopathology domains of schizophrenia are summarized in No add-on RCTs with new novel antidepressants vortioxetine, vilazodone, or agomelatine could be located.The majority of the included studies reported overall good tolerability of antidepressant-antipsychotic combinations. We were able to identify 36 RCTs on combinations of antidepressants with an ongoing antipsychotic treatment published in the period from 1968 to 2013. The RCTs comprised the majority of the existing antidepressants and aimed to study their efficacy for the main clinical domains of schizophrenia. To summarize, the existing evidence regarding the efficacy of antidepressants in the treatment of schizophrenia is mixed.The methodological quality of the studies varied. Overall, the later RCTs reported more precisely than the earlier ones the outcome criteria, the dosages of the study drugs, the clinical scales used, the patient population’s clinical and demographic characteristics, etc. This progress of the clinical study methodology advocates, in general, giving more weight to the most recent studies. Nevertheless, both old and recent RCTs have a number of restrictions. Almost all of them comprise small patient samples. Many publications express secondary analysis from the studies originally designed for negative  but not affective symptoms. This uncertainty possibly contributes to the heterogeneity of results obtained in different studies and makes a between-study comparison difficult.Only 5 of 14 RCTs with SSRIs reported favorable results. This puts into question the efficacy of this antidepressant group in the treatment of schizophrenia’s negative symptoms. From a theoretical viewpoint, these findings possibly indicate that the increase of serotonin availability in the brain per se is not enough to alleviate the negative symptoms.The data on selective serotonin and noradrenaline reuptake inhibitor (SNRIs) are scarce. Only one small study (with duloxetine) is available. Its desirable result needs further validation in larger studies. Studies with other SNRIs are also necessary.For the TCAs, even though 2 of the 3 existing studies were positive, the small number of trials and methodological issues  makes the significance of these results uncertain.In both available studies with reboxetine and bupropion, the active drug was not more efficacious than placebo, indicating that catecholamine reuptake inhibition alone is seemingly insufficient to alleviate negative symptoms of schizophrenia.Data on the receptor-blocking antidepressants are more convincing, since they outperformed placebo in 10 of the 13 reported RCTs. This advantage may plausibly be interpreted with the dissimilar pharmacodynamic mechanisms of action of these compounds. While not affecting monoamine transporters (and thus reuptake of monoamines), all receptor-blocking antidepressants inhibit the postsynaptic serotonin 5HT2 receptors, a property that they share with clozapine and other SGAs . The theOn the other hand, in 4 of 6 positive studies, mirtazapine was added to risperidone, clozapine, or olanzapine –each of them known to be a potent 5HT2A/C blocker by itself, which makes little sense from the viewpoint of the above-mentioned atypicality theory. A possible explanation for this advantageous effect may be the affinity of mirtazapine to some other (than 5HT2) types of receptors. In addition to the 5HT2 receptors, mirtazapine inhibits the postsynaptic serotonin 5HT3 receptors and presynaptic noradrenaline α2 receptors and indirectly stimulates postsynaptic serotonin 5HT1A receptors , all of Blockade of the noradrenaline α2 receptors also may be useful in the treatment of schizophrenia. In an RCT by Rummel et al. (2005) performed a systematic review of 5 RCTs  available at that time and fulfilling the rigorous systematic review criteria. The authors concluded that “the combination of antipsychotics and antidepressants may be more effective in treating negative symptoms of schizophrenia than antipsychotics alone.” This conclusion, although not differentiating between effects of different antidepressant subgroups, is mainly in line with that of the current review.The efficacy of add-on antidepressants on negative symptoms of schizophrenia was reviewed by Antidepressants seem to have no desirable effect on positive symptoms of schizophrenia, as almost all RCTs were disappointing in this respect. The only exception was a study by In clinical practice, physicians are sometimes concerned with a risk of worsening or exacerbation of psychosis when prescribing antidepressants to patients with schizophrenia . MoreoveOnly 2 of 6 studies with SSRIs were positive, and exactly the same proportion (2 of 6) was found with regard to receptor-blocking antidepressants. This puts the efficacy of both antidepressant groups into question. The antidepressants with sound noradrenaline activity may, however, be of interest for this symptom domain, since 1 of the 2 existing RCTs with reboxetine, the only study with imipramine, and the only study with duloxetine  reported improved depression. A trend in favor of active compound over placebo was observed in 1 of 2 RCTs with adjunctive noradrenaline and dopamine inhibitor bupropion. Of the 6 published trials with the receptor-blocking antidepressants, only one  reportedTo summarize, the evidence regarding the efficacy of antidepressants in the treatment of depression in schizophrenia is scarce and controversial. The main methodological issue in this area is that the study population was not selected specifically for depressive symptoms, with the only exception of the postpsychotic depression study by Also, For the monoamine transporter inhibitors, only SSRIs and the noradrenaline reuptake inhibitor reboxetine studies were identified. All these studies were negative, probably not surprisingly given the lack of theoretical basis for such combination in EPS. Moreover, the SSRIs are reported to even cause EPS in patients with major depressive disorder .For the receptor-blocking antidepressants, a positive effect on the FGA-induced EPS was found in both studies with trazodone  but in nThe use of receptor-blocking antidepressants for the EPS arises from the theoretical assumption of dopamine insufficiency in the basal ganglia as the cause of EPS . InhibitIn sum, despite encouraging theoretical presumptions, the evidence regarding the efficacy of receptor-blocking antidepressants for antipsychotic-induced EPS is limited and contradictory. Only trazodone and nefazodone were found effective, while mianserin and mirtazapine were probably not. As marketing of nefazodone has been discontinued, the nefazodone-related findings are no longer relevant for clinical practice. Moreover, positive findings with receptor-blocking antidepressants are available for only FGA-induced EPS and may be extrapolated on the SGAs only with caution. Given that the use of FGAs has been markedly diminished during recent years and further decrease may happen in the future, the results of these studies are rather of theoretical than of practical interest.Neurocognitive effects of antidepressants added to antipsychotics in schizophrenia have been studied in only 3 RCTs.A serotonin reuptake inhibitor, citalopram, did not improve cognition in schizophrenia . The incOn the contrary, 2 receptor-blocking antidepressants with a similar mechanism of action, mianserin  and mirtAs for the negative symptoms, the plausible proneurocognitive effect of mirtazapine and mianserin in schizophrenia may emerge from their receptor-binding profile. First, they may enhance prefrontal catecholamine activity via 5HT2A or 5HT2C receptor blockade (as most SGAs do), thereby enhancing activity of the prefrontal cortex . Second,The number of studies presented in both reviews was small. Thus, the evidence for the proneurocognitive potential of add-on antidepressants is, so far, insufficient but deserves further research.The RCT is the gold standard of clinical efficacy studies. However, RCTs are fraught with patient selection bias and, thus, the results of available naturalistic prospective studies, mirror design studies, as well as pertinent register studies should be taken into account for balanced clinical decisions. Furthermore, we have not performed quantitative analyses of the data nor included unpublished data as it would be required for a systematic meta-analysis. Moreover, the vast majority of the available RCTs are small and methodologically heterogenic, which calls for caution when interpreting our results. In a substantial part of the RCTs in this review, the reported outcomes were a result of secondary analysis.This review has focused predominantly on the efficacy of adjunctive antidepressants, while in clinical practice the potential benefits should be balanced against unwanted side-effects of these drugs and of antidepressant-antipsychotic combinations.In the future, well-designed, larger, and longer RCTs are needed. There is also a need for trials designed specifically for certain symptom domains, with appropriately selected patients and pertinent rating scales. Despite an existing theoretical rationale, there is a lack of studies assessing the effects of bupropion, reboxetine, SNRIs, and reversible inhibitors of monoamine oxidase A on cognitive function in schizophrenia.The future studies should also focus on new, novel, add-on antidepressants that have come into clinical practice in recent years. Of interest may be, for instance, vortioxetine. In addition to the conventional inhibition of the serotonin (5HT) transporter, vortioxetine demonstrates an affinity to the serotonin 5HT1a, 5HT1b, 5HT1d, 5HT3, and 5HT7 receptors. Furthermore, it modulates the GABA, MNDA, noradrenergic, dopaminergic, cholinergic, and histaminergic neurotransmitter systems . Some ofThere exists a substantial number of RCTs on the efficacy of add-on antidepressants, but due to methodological issues the results remain uncertain. Receptor-blocking antidepressants mirtazapine and mianserin show somewhat consistent efficaciousness for negative symptoms. The evidence for EPS, neurocognition, and depression domains is even more ambiguous and should be interpreted with caution. Some receptor-blocking antidepressants (trazodone and nefazodone) seem to improve FGA-induced EPS, while others (mirtazapine and mianserin) appear to enhance neurocognition. Transporter-blocking antidepressants with prominent noradrenergic activity tended to improve depressive symptoms.The overall data for the SSRIs are rather disappointing. An add-on antidepressant (mirtazapine) improved positive symptoms in only one study. However, the data unequivocally show that adjunctive antidepressants do not worsen psychosis, at least in chronic schizophrenia. The caution recommended for antidepressants in some current schizophrenia treatment guidelines may, therefore, need reappraisal.None.


Cyclic AMP is a ubiquitous intracellular second messenger involved in the regulation of a wide variety of cellular processes, a majority of which act through the cAMP – protein kinase A (PKA) signaling pathway and involve PKA phosphorylation of specific substrates. PKA phosphorylation events are typically spatially restricted and temporally well controlled. A-kinase anchoring proteins (AKAPs) directly bind PKA and recruit it to specific subcellular loci targeting the kinase activity toward particular substrates, and thereby provide discrete spatiotemporal control of downstream phosphorylation events. AKAPs also scaffold other signaling molecules into multi-protein complexes that function as crossroads between different signaling pathways. Targeting AKAP coordinated protein complexes with high-affinity peptidomimetics or small molecules to tease apart distinct protein–protein interactions (PPIs) therefore offers important means to disrupt binding of specific components of the complex to better understand the molecular mechanisms involved in the function of individual signalosomes and their pathophysiological role. Furthermore, development of novel classes of small molecules involved in displacement of AKAP-bound signal molecules is now emerging. Here, we will focus on mechanisms for targeting PPI, disruptors that modulate downstream cAMP signaling and their role, especially in the heart. Figure 1) governed by protein–protein interactions (PPIs) creates an opportunity for new therapeutic strategies to control cAMP dependent signaling that is out of tune or involved in pathologies. In this review we will first focus on signaling through AKAP-coordinated complexes, next on targeting PPIs as a possible strategy to control and regulate cAMP signaling events and finally mention a few examples of possible PPIs that could be targeted. We will discuss cAMP/PKA/AKAP signaling in general terms but will particularly focus on the heart, where cAMP signaling pathways are involved in different stages of the cardiac cycle and in several pathologies.Intracellular 3′-5′-cyclic adenosine monophosphate (cAMP) is an important second messenger that regulates a number of biological processes. Even though cAMP is diffusible, its concentration and signaling are tightly controlled and coordinated through the involvement of a molecular machinery coordinating the spatial and temporal processes of localized cAMP signaling events. Signal transduction through the cAMP pathway starts by stimulation of G-protein-coupled-receptors (GPCRs), via specific extracellular ligands leading to activation of adenylyl cyclase (AC), which converts ATP into cAMP. The rise in intracellular cAMP levels leads to a set of events mediated by specific effector molecules, hereunder protein kinase A PKA; , cyclic Cyclic AMP is a ubiquitous intracellular second messenger involved in the regulation of a wide variety of cellular processes. For this reason there are molecular mechanisms that rigorously control cAMP signal responses, intimately linked with spatial and temporal fine tuning. The first indication that cAMP could be organized in specific microdomains came in the early 1980s, when different GPCR ligands were observed to trigger very different cardiomyocytes responses, even if there was a similar increase in cAMP levels . MeanwhiFirst, an important starting point for generation of well-defined local gradients of cAMP is the organization and diverse expression of receptors, G proteins and associated cyclases at the plasma membrane. The G proteins are heterotrimeric, guanosine triphosphate-binding (GTP-binding) proteins, assembled from three subunits: α-, β- and γ-. There are more than 20 different Gα-subunits that can activate or inhibit different effectors. For example, Gαs activates and Gαi inhibits ACs, respectively. Like G proteins, AC exist in different isoforms in mammals, most of them are associated with the plasma membrane (AC1-9) whereas AC10 is soluble . Both G 2+ levels, while PKA modulates contractility via an increase in intracellular Ca2+ . PDEs are highly conserved between species, with around 50 different isoforms that are part of 11 families (PDE1-11) with different enzymatic and regulatory characteristics . They alFourth, scaffold proteins coordinate the physical assembly of components of a signaling pathway. In cAMP signaling pathways AKAPs are responsible for the assembly of specific signalosomes. They form complexes between PKAs and their specific targets in localized subcellular domains, consequently they are important contributors to compartmentation, which will be further discussed in the next chapter.Figure 2) are:The spatial and temporal organization of cAMP/PKA signaling is attained by a carefully tuned balance between local activation of the signal effector and signal termination machinery assembled and targeted by AKAPs. There are more than 50 AKAPs identified, and even though they belong to a structurally diverse family they all share the ability to enable tightly regulated phosphorylation of substrates that are anchored to or localized in the vicinity of AKAPs together with PKA . The fou(1) Anchoring of the dimer R-subunit D/D-domain through interaction between the hydrophobic pocket of PKA and the 14–18 amino acid amphipathic helix region of AKAPs . AKAPs c(2) Targeting to specific subcellular locations. AKAPs typically contain a targeting domain that localizes the entire AKAP/PKA complex inside the cell. This can be a defined organelle, membrane or structure and the binding may occur by protein–protein or protein–lipid interactions.(3) Directly binding to or co-localizing with specific substrates that will be phosphorylated by PKA.(4) Assembly of multi-protein complexes with additional signaling enzymes such as PDEs, phosphatases (PPs), and other kinases;AKAPs can be found in a number of tissues and are involved in the composition of a wide variety of complexes implicated in different signaling cascades . Small interfering RNAs (siRNAs) can be specifically designed to target any gene and can silence target mRNA expression to overcome different pathologies. Even though siRNA is been increasingly used and some siRNA based therapies are in human clinical trials, siRNA therapeutics still need to overcome the obstacles of efficient drug delivery to be a fully viable drug development strategy  can, when linked to the disruptor peptide, easily facilitate its transport into the cell. TAT-AKAP-IS could at micromolar concentrations disrupt endogenous AKAP/PKA interaction and affect PKA subcellular localization in insulin-secreting pancreatic B-cells  domain that binds to AKAP18δ and competes with and displaces the AKAP18δ/PLB interaction . This PLSelect , thermal shift assay, surface plasmon resonance (SPR), immunoprecipitation and other biochemical assays as well as in silico modeling of interactions. However, there are still challenges when screening for small molecule PPI disruptors: the PPI interfaces may be big, discontinuous or flat and hydrophobic with an absence of pockets and typically a screen yields PPI disruptors with micromolar affinity. Peptides do not have these limitations, but have issues with stability and permeability. In order for small molecule targeting of PPIs to be successful, a topology of the interacting proteins with small pockets or the identification of key residues that contribute to the binding energy, so called “hot spots” are necessary requirements  for compounds targeting PPIs has resulted in an increasing number of projects in this area. Combinations of primary and secondary assays in HTS can create an attractive and very useful setting to screen fast and easily large compounds libraries. These are normally miniaturized assays performed in a robotics workstation, where different instruments and liquid handling systems are used. There are several published assays to screen both for peptides and small molecules that were specifically developed to target PPIs in complexes involving AKAPs such as AlphaScreen; SPR; enzyme-linked immunosorbent assay (ELISA); and homogenous time-resolved fluorescence (HTRF) .Certainly targeting PPIs is a very appealing and promising therapeutic strategy as it is possible to specifically target interactions in single molecules complexes at defined subcellular places that until now has not been fully address and exploited.2+-handling and excitation-contraction coupling; (ii) hypertrophic stress responses; and (iii) controlling electrical signaling. Here we will shortly mention a few of the most relevant AKAPs targets.Heart pathologies are a leading cause of hospitalization and mortality in the Western World. Moreover, population growth and increase in life expectancy accelerates the number of heart incidents. Currently there are several treatment choices that interfere with cAMP signaling in the heart. Beta-blockers block adrenergic signaling and have a negative inotropic and chronotropic effect. In contrast dopamine has a positive inotropic effect and paces the heart. Beta-blockers are, however, accompanied by significant side effects, because they affect all downstream signaling. As an alternative to beta-blockers, the recently described biased agonists and antagonists can provide functional selectivity, where the biased ligand activates or terminates a specific intracellular signaling pathway downstream of the GPCR. In this case one can achieve a more specific and selective effect rather than the “all or nothing” effect of beta-blockers . Particu2+ channels in plasma membrane. It was also shown that in skeletal muscle cells AKAP18α directly interacts with both PKA and the channel  anchors PKA to L-type Ca channel . The aut2+ channels together with β-adrenergic receptor, PKA, AC5/6 and calcineurin (CaN), which is important for sympathetic regulation  assembles a complex in caveolin 3-associated L-type Cagulation . Also, A AKAP18α . Taken t2+-ATPase 2 (SERCA2) in the heart, which was also later shown in human myocardium , 2+- re-uptake into the SR. Also, removal/reduction of AKAP18δ by siRNA injected in adult cardiomyocytes had the same effect.ocardium . The PLB, CaN and transcription factors NFAT and MEF2 , 2013. Ies mAKAP , 2013. Aes mAKAP .In vivo evidence showed that mAKAP knockout mice have better chance of survival when cardiac hypertrophy is induced both by pressure and catecholamine overload. It was also shown that mAKAP knockout decreased apoptosis, fibrosis and pathological gene expression via decreasing activation/phosphorylation of PLC∈/PKD1/HDAC4 complex proteins   .AKAP-Lbc acts as a scaffold protein for several protein kinases: PKA, protein kinase C (PKC) and PKD that phosphorylate different substrates leading to hypertrophy, additionally AKAP-Lbc can also acts as a guanine nucleotide exchange factor (GEF) for the small GTPase Rho  in the heart . HSPs arAs previously mentioned AKAP-Lbc also recruits PKC and PKD1 which enables phosphorylation and activation of PKD1 by PKC , the actin vivo role of AKAP in regulating cardiac hypertrophy .A-kinase anchoring proteins also play a role in promoting electrical cell-to-cell coupling. Gap junctions play a crucial role in cell-to-cell conductance in cardiomyocytes. These channels are composed of connexin 43 (Cx43) hexamers creating pores throught the cell membrane of two adjacent cells allowing passage of the signal. It has recently been shown that an AKAP, ezrin, is involved in the expression and regulation of gap junction conductivity by organizing a PKA/Cx43/ezrin supramolecular complex in other cell types . MoreoveEvidence that cAMP compartmentation is particularly important for transmission of accurate and specific biological information is increasing. In order to respond to local cAMP gradients AKAPs contribute to correct and specific propagation of the cAMP signal by organizing supramolecular complexes where PKA, its substrates and other signaling proteins are assembled. These complexes come together through PPIs, which are fundamental for correct propagation of the response. Until recently targeting AKAP interactions by designing peptides to disrupt such binding interfaces was predominantly used as a tool to study function of components of these signalosomes. Currently, there is growing interest in the possibility to develop small molecule PPI disruptors which creates new opportunities for developing therapeutic strategies by preventing interactions in AKAP-complexes. Here we suggest some possible target complexes in the heart, however, other potential targets were not mentioned and more may still be discovered. Finally, we expect that targeting PPIs in complexes organized by AKAPs will receive increased attention as knowledge in development of small molecule PPI disruptors increases and the benefits of specifically perturbing individual signaling complexes come out.The authors do research on small molecule PPI disruptors for perturbing AKAP complexes and have patent applications in this area that could constitute future commercial interest. There are no financial relationships that could be construed as a potential conflict of interest.


The HTLV-1 gp21 glycoprotein is involved in envelope trafficking and membrane targeting while bZIP protein is indispensable for cell growth and proliferation. This study aimed to assess the molecular diversity of gp21 and HBZ proteins, in TSP/HAM and healthy carriers. DNA samples from HTLV-1 infected individuals were submitted to PCR, sequencing, and the molecular analyses were performed using bioinformatics tools. From eight gp21 analyzed sequences one amino acid change (Y477H) was associated to the switch of helix to coil structure at secondary structure prediction. From ten HBZ analyzed sequences, two amino acid changes were identified (S9P and T95I) at the activation domain. One mutation (R112C) located at nuclear localization signal was present in 66.7% and 25% of HC and TSP/HAM groups, respectively. This is the first report of mutations in HBZ region. These polymorphisms might be important for viral fitness.


Human T-lymphotropic virus type 1 (HTLV-1) has worldwide distribution and is considered endemic in many world regions, including southwestern Japan and Brazil. Japanese immigrants and their descendants have a high risk of acquiring this infection due to intense population exchange between Brazil and Japan.This cross-sectional study aimed to estimate the prevalence of HTLV, analyze the main risk factors associated with this infection, identify the main circulating types and subtypes of HTLV in Japanese immigrants and descendants living in Campo Grande-MS (Middle-West Brazil), as well as analyze the phylogenetic relationship among isolates of HTLV.A total of 219 individuals were interviewed and submitted to blood collection. All collected blood samples were submitted for detection of anti-HTLV-1/2 using the immunoassay ELISA and confirmed by immunoblot method. The proviral DNA of the 14 samples HTLV- 1 positive were genotyped by nucleotide sequencing.The overall prevalence of HTLV-1 was 6.8% . Descriptive analysis of behavioral risk factors showed statistical association between HTLV-1 and age greater than or equal to 45 years. The proviral DNA of HTLV-1 was detected in all HTLV-1 positive samples. Of these, 14 were sequenced and classified as Cosmopolitan subtype, and 50% (7/14) belonged to subgroup A  and 50% (7/14) to the subgroup B (Japanese).The high prevalence of HTLV-1 found evidence of the importance of early diagnosis and counseling of individuals infected with HTLV-1 for the control and prevention of the spread of this infection among Japanese immigrants and their descendants in Central Brazil. The population of Okinawan immigrants is considered vulnerable to human T-lymphotropic virus type 1 (HTLV-1) infection because the Okinawa region in Japan is an endemic area. The second Brazilian largest Okinawan community is set in Campo Grande, Middle-West Brazil. This study aimed to estimate the prevalence and risk factors associated with HTLV infection among Japanese immigrants and their descendants living in Campo Grande. The prevalence of 6.8% of HTLV-1 infection that was found is considered high. The HTLV-1 infection was associated with age ranged from 45 years old or older. Most infected individuals are Okinawan descendants. The HTLV-1 rate found in the present study indicates that the prevalence of this infection remains high among this Japanese community. This study emphasizes the importance of implementing preventive and diagnostic public health policies to decrease the risk of HTLV-1 transmission among Japanese communities throughout Brazil. The retrovirus human T-lymphotropic virus type 1 (HTLV-1) is associated with many severe diseases, including adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), but most infected people remain asymptomatic .Approximately 10 million people are estimated to be infected with HTLV-1 throughout the world . A high Since confirmatory tests for the diagnosis of HTLV infection were not available in the 80s, the prevalence found by Kitagawa and cols (1986) could be overestimated due to the presence of false positives. Therefore, considering the lack of regional studies on HTLV infection, the Japanese immigration wave to Brazil, particularly to Mato Grosso do Sul state and the risk of intrafamilial transmission of HTLV-1, the main goal of this study was to revisit the situation of HTLV-1 epidemiology, especially its estimated prevalence and molecular characterization around 30 years after the first published epidemiological study in Japanese immigrants and their descendants living in Campo Grande, MS.This cross-sectional study was conducted between April 2012 and October 2013 in the city of Campo Grande, capital of the state of Mato Grosso do Sul in midwestern Brazil, which has about 786,797 inhabitants with an estimated contigent of 20,000 Japanese-Brazilian descendants from 4,000 families, showing high migration of this population to Campo Grande [In order to get a statistical power of 80% (β = 20%), a significance level of 95%(α <0.05) and an accuracy of 4%, the calculated sample size to estimate the prevalence of HTLV-1 infection, should include at least 216 individuals, based on the prevalence of 10.0% for anti-HTLV-1/2 found by Kitagawa et al. (1986). Two hundred and twenty two potential subjects met the sample criteria. Of them, 219 (88.6%) agreed to participate. Therefore, the study population included 219 participants, who were from, approximately, 70 families in a population of about 18,000 Japanese immigrants and Japanese descendants living in Campo Grande-MS. Individuals were eligible to participate if they were older than 18 years of age, Japanese immigrant (born in Japan), Japanese descendant (had any genetic relationship to a Japanese immigrant) and non-descendant (had relationship to a Japanese immigrant only by marriage) and able to provide written informed consent. Pregnant women were excluded from the study. The presence of other illness or other infections was not used as selection criteria. The participants were previously advised about the campaign through posters and leaflets placed in the Okinawa Association and Brazilian Nipo Association. In addition to anti-HTLV test, free testing for diabetes and blood pressure were offered by the health campaign. The participants did not know their serological profile for anti-HTLV-1/2.They were informed of the objectives and methods of the study and written informed consent was obtained from all study participants prior to enrollment. Following enrollment, a questionnaire was administered to obtain sociodemographic characteristics and identify risk factors associated with HTLV transmission, including age, gender, immigrant generation, history of residence in Japan, history of blood transfusion, surgery, personal item/sharps sharing, injection drug use, breastfeeding, multiple partners, unprotected sex and sexually transmitted diseases.® Genomic, Promega, Madison, WI, USA), according to manufacturer’s instructions. Nested PCR amplified the targeted portion of the 5’LTR of HTLV-I. Reactions were performed as previously described [TM PCR DNA and Gel Band Purification Kit  according to the manufacturer’s instructions. The fragments were sequenced using BigDye Terminator Cycle Sequencing Ready reaction Kit and ABI 1373 . Nucleotide sequences were aligned and compared with published HTLV-1 sequences from various geographic regions using ClustalX and Mega 5.05. Philogenetic trees were constructed by use of the neighbor-joining (NJ) method. The NJ tree was evaluated by bootstrap analyses of 1000 replicates.Blood samples were collected from study participants and serum samples were evaluated by an enzyme-linked immunosorbent assay (ELISA) test for the presence of anti-HTLV 1 and 2 antibodies  and confirmed by Western Blot (WB) assay with a commercial test (MP Diagnostics HTLV BLOT 2.4–Singapore). The samples reactive by screening and positive by WB were considered positive for HTLV-1 or 2 infections. DNA was extracted from whole blood samples using the DNA Genomic Purification Kit , a statistical software package. Statistical analyses were performed using SPSS Statistics Data Editor . HTLV prevalence was estimated using 95% confidence intervals (95% CI). Potential risk factors for HTLV infection were assessed in univariate analysis. Chi-square and Fisher’s exact tests were performed for comparison of categorical parameters. Chi-square and Fisher tests were two-tailed. Statistical significance was considered at p≤0.05.The present study was approved by the Ethics Committee on Research Involving Human Subjects of the Federal University of Mato Grosso do Sul (CEP/UFMS), under protocol number 2249 CAAE 0329.0.0.049.000–11. All adult subjects provided written informed consent, and a parent or guardian of any child participant provided written informed consent on their behalf.A total of 219 individuals, Japanese immigrants or descendants and non-descendants of Japanese immigrants living in Campo Grande-MS were enrolled in this study. Most of those were female (61.2%). Mean age was 54.4 years (range 11 to 101 years) and the majority (69.9%) had 45 years of age or older.The majority (88.1%) was born in Brazil and 10.9% were born in different regions of Japan  and 1.0% was from Japanese descendants from Peru. The Brazilian group (n = 193) was composed of 187 (96.8%) Japanese descendants and 6 (3.1%) non-Japanese descendants. The majority (54.8%) of participants reported having a steady sexual partner (married) or cohabitating with a sex partner. Regarding education, 74% had completed secondary education or college.The prevalence of HTLV-1 infection was 6.8% (95% IC: 3.5 to 10.2). None of the 219 individuals presented anti-HTLV-2 antibodies. None of the positive subjects showed clinical signs compatible with HAM/TSP or ATL, and therefore, were classified as asymptomatic. The frequency of anti-HTLV seropositivity was higher among Okinawan descendants (8.1% vs. 2.1%) than among descendants from other regions of Japan and non-Japanese descendants; however, this difference was not statistically significant.Among studied population, a frequency of 6.1% of HTLV-1 infection was found among Japanese-Brazilian descendants participants, while 12.5% was observed in those who declared themselves as foreign, which comprise 22 Japanese and 2 Peruvian nationals. Most infected subjects (n = 14) claimed to be Okinawan descendant, and only one who was not a descendant was positive for anti-HTLV.P = 0.04). The mean age of subjects positive for HTLV-1 was 70 years, which was greater than that of 53 years for non-reactive subjects. Risk factors for HTLV infection among the studied population are shown in The proviral DNA of HTLV-1 was detected by amplification of the 5 'LTR region by nested PCR in all samples (n = 15) HTLV-1 positive. Of them, 14 were sequenced and classified as Cosmopolitan subtype and 50% (7/14) belonged to subgroup A  and 50% (7 / 14) to the subgroup B (Japanese). The new nucleotide sequence samples described in this study have been deposited in GenBank with accession number as follows: OKW21 (KM023750), OKW24 (KM023751), OKW46 (KM023752), OKW63 (KM023753), OKW72 (KM023764), OKW84 (KM023754), OKW107 (KM023761), OKW112 (KM023755), OKW131 (KM023756), OKW151 (KM023757), OKW152 (KM023758), OKW165 (KM023759), OKW209 (KM023760), OKW235(KM023767) were compared with nucleotide sequences of 17 isolates of HTLV-1 available from GenBank . The GenCurrently, there are approximately an estimated 1.5 million Japanese descendants living in Brazil, whose vast majority reside in the state of São Paulo (Southwest region), followed by the state of Paraná (South region) and Mato Grosso do Sul . In Brazil, 10% of the descendants of Japanese immigrants originate from the province of Okinawa, and the second largest Okinawan community in Brazil is in Mato Grosso do Sul State .The population of Okinawan immigrants is considered at risk of acquiring HTLV-1 due to originating from the endemic islands of Okinawa, Japan, where the prevalence of HTLV-1 varies between 14% and 31% [In the present study, 6.8% HTLV-1 infection prevalence was observed. This percentage is considered high, indicating there should be greater attention given to HTLV-related diseases in this population. This prevalence is 40 and 52 times higher than those found in prime blood donors (0.17%) and pregnant women 0.13%) from Campo Grande , respectively, and 13.6 times higher than that found in African descendants residing in Central Brazil (0.5%) ,11,12. I% from CaP = 0.28) [When comparing the prevalence of HTLV-1 among members of the Japanese community of Campo Grande in the year of 1986 with the current prevalence found in this study, we observed the maintenance of high prevalence over the last 27 years (10% vs 6.8%). This difference was not significant ( = 0.28) . Maybe,  = 0.28) . In addi = 0.28) .Increasing age was significantly associated with HTLV-1 infection. This finding was also reported in previous studies and may be due to the cumulative risk of HTLV-1 infection over the lifetime of individuals surveyed ,16. The Although breastfeeding is known to play an important role in the transmission of HTLV-1, it was not statistically significant in our study ,24. In 2All of HTLV-1 isolates were classified as Cosmopolitan subtype (HTLV-1a) and half of them (50%) belonged to subgroup A  and 50% to subgroup B (Japanese). In Brazil, the Cosmopolitan is the most prevalent subtype of HTLV-1 and the Transcontinental subgroup (HTLV-1aA) is the most prevalent among them, followed by the Japanese subgroup (HTLV-1aB), which is detected among Japanese-descendants and immigrants, such as in the present study . In JapaAll individuals who are positive for HTLV-1 infection in this study were asymptomatic, and therefore, mostly likely unaware of their serological profile. Thus, the individual may remain asymptomatic and continue to infect sexual partners and family . ConsideThe HTLV-1 prevalence of 6.8% (95% CI: 3.5 to 10.2) found in the present study indicates the maintenance of high prevalence of this infection in the Japanese community of Campo Grande-MS over the past 27 years. These findings also showed that the prevalence of HTLV-1 infection is distinct among regions of Brazil, and although high, this rate did not contribute to an increase in HTLV-1 infection prevalence among the general Brazilian population. Further HTLV-1 nucleotide sequencing may provide more information on the molecular epidemiology of this infection in Japanese descendants population living in Brazil, which may be helpful in understanding the HTLV-1 transmission in these isolated communities.S1 Checklist(PDF)Click here for additional data file.


Human T-lymphotropic virus type 1 (HTLV-1) infects an estimated 10 million persons globally with transmission resulting in lifelong infection. Disease, linked to high proviral load, occurs in a minority. In established infection HTLV-1 replicates through infectious spread and clonal expansion of infected lymphocytes. Little is known about acute HTLV-1 infection. The kinetics of early HTLV-1 infection, following transplantation-acquired infection in three recipients from one HTLV-1 infected donor, is reported. The recipients were treated with two HTLV-1 enzyme inhibitors 3 weeks post exposure following the detection of HTLV-1 provirus at low level in each recipient. HTLV-1 infection was serially monitored by serology, quantification of proviral load and HTLV-1 2LTR DNA circles and by HTLV-1 unique integration site analysis.HTLV-1 antibodies were first detected 16–39 days post-transplantation. HTLV-1 provirus was detected by PCR on day 16–23 and increased by 2–3 log by day 38–45 with a peak proviral doubling time of 1.4 days, after whichsteady state was reached. The rapid proviral load expansion was associated with high frequency of HTLV-1 2LTR DNA circles. The number of HTLV-1 unique integration sites was high compared with established HTLV-1 infection. Clonal expansion of infected cells was detected as early as day 37 with high initial oligoclonality index, consistent with early mitotic proliferation.In recipients infected through organ transplantation HTLV-1 disseminated rapidly despite early anti-HTLV-1 treatment. Proviral load set point was reached within 6 weeks. Seroconversion was not delayed. Unique integration site analysis and HTLV-1 2LTR DNA circles indicated early clonal expansion and high rate of infectious spread. Human T-lymphotropic virus type-1 (HTLV-1) is associated with two main clinical disorders that arise in 5–8 % of carriers: HTLV-1 associated myelopathy (HAM), a progressive, inflammatory, spastic paraparesis, and adult T cell leukaemia/lymphoma (ATLL), an aggressive malignancy of CD4+ T-lymphocytes. The majority of infection occurs through sexual intercourse or from mother-to-child during breast feeding but infection from unscreened blood transfusions or organ transplants can occur . KnowledEvidence from phylogenetic studies and integration site analysis reveals two routes by which HTLV-1 propagates within the host –16: infeThe relative contributions of infectious and mitotic spread at different time points of infection are not known; this knowledge is required for designing rational treatment protocols. HTLV-1 viral RNA is rarely found in human plasma , 22, whiSince late 2002, all blood donations in the UK have been screened for HTLV-1  but realThe liver and both kidneys were retrieved from a deceased female Caucasian donor, who was not known to carry, and had no risk factors for, HTLV-1 infection. The organs were transplanted in accordance with UK Blood and Transplant service protocols to three HLA class-matched male recipients. At the time of organ retrieval the donor HTLV status was reported as ‘awaited’ but after transplantation the HTLV-1 seropositive status of the donor was detected and confirmed, following which the recipients were informed. No suitable samples for quantifying the proviral load of the donor were available.Clinical details of the recipients are summarised in Table HTLV-1 infection was diagnosed by HTLV-1 DNA PCR in all three recipients who were then commenced on zidovudine and raltegravir, which inhibit HTLV-1 reverse transcriptase , 26 and In case 1 the first detection of anti-HTLV-1 antibodies was 16 days post-transplantation. The enzyme linked immunoassay (EIA) sample/cut-off (S/CO) optical density was 6.88 and faint bands indicative of antibodies to p24 (gag), rgp46-1 (env) and anti-GD21 (env) were seen on the western blot  in any of the recipients (<0.01 % PBMC infected). However, by nested PCR provirus was detectable and estimated at 0.003 % PBMC in Case 1 (liver transplant) and at 0.01 % in Case 2 (kidney transplant) but remained undetectable in Case 3 (kidney transplant) until day 23 when it became detectable at 0.01 % by qPCR. HTLV-1 proviral load increased in each case by 2–3 logs between days 16–23 and days 38–45, after which a steady state was reached with about 1 % PBMCs infected. Since it is known that there is a single copy of HTLV-1 integrated into each infected cell , the pro−5 2LTR DNA circles/infected cell) and plateaued from day 45 (3.6 × 10−6 2LTR DNA circles/infected cell).In case 1: 2LTR DNA circles were measured at five time points . They were undetectable at day 16, peaked at day 31 (4.2 × 10−3 2LTR DNA circles/infected cell) and continued to decline .In case 2: 2LTR DNA circles were measured at four time points . They were undetectable at day 16, peaked at day 38 (0.21 2LTR DNA circles/infected cell) and declined by the next time point at day 93 (3.3 × 10−3 2LTR DNA circles/infected cell) which had declined by next time point  followed by a second peak at day 142 (0.16 2LTR DNA circles/infected cell) coinciding with a peak in the proviral load.In case 3: 2LTR DNA circles were measured at seven time points  and were undetectable at day 16, with an initial peak at day 36  were detected and the oligoclonality index was high; both these parameters subsequently decreased (Panel C). In this case the peak proviral load may be attributed to both an increase in the absolute number of integration sites (suggesting infectious spread) and vigorous expansion of a small number of clones.Case 3: The oligoclonality index, reflecting clonal proliferation, peaked at day 48 and then declined over the next 3 months, with an increase in the absolute number of UIS consistent with infectious spread (Panel C).5) in these transplant recipients was substantially higher than those previously obtained in asymptomatic carriers (median 9.0 × 103) [4) [3) [The number of clones was estimated from the integration site high throughput sequencing data using a newly developed and published method, DivE. DivE compares multiple mathematical models fitted to rarefaction curves, which give the expected number of infected clones as a function of the number of infected cells. The estimated number of clones (median 1.3 × 100 × 103) , patient103) [4) , and pat [4) [3) .For the majority of individuals, HTLV-1 is transmitted from mother-to-child via breast feeding and as such it is nearly impossible to conduct a study during acute infection. This report of an unfortunate clinical event has provided a rare opportunity to quantify the kinetics of acute HTLV-1 infection. We report that anti-HTLV-1 antibody responses were not delayed by immunosuppressive therapy: Anti-HTLV antibodies were detected in two cases at the first available time point (day 16) and in the third by day 39. This is similar to reports from recipients of HTLV-1-infected blood transfusions, in whom the seroconversion window has been estimated at 55 days , with 50We report that HTLV-1 spread early and rapidly in the three transplant recipients. In established HTLV-1 infection the proviral load varies more than 5 logs between individuals, and a high proviral load predicts and predates HTLV-1-associated disease. Proviral load remains stable within an individual over many years –34 but iEarly HTLV-1 spread is both ‘infectious’ and ‘mitotic’. We observed that the HTLV-1 2LTR DNA circles peaked with the peak proviral load, and both declined by the next testing time point. 2LTR DNA circles have not been previously reported in HTLV-1 infection and thus there are no data on the survival of such episomal viral DNA in HTLV-1 infection. The assumption that these 2LTR DNA circles are markers of recent infection is based upon this phenomenon in HIV . Here, wIn each case, the peak oligoclonality index also coincided with the peak proviral load. The oligoclonality index reflects the degree of mitotic proliferation, which in case 2 at day 39  according to the manufacturer’s protocols.HTLV-1 antibodies were detected by Murex HTLV I + II  and confirmed by Western blot using Genelabs Diagnosticstax and human β-globin gene, assuming one copy of tax [β-globin per infected cell. Samples with unquantifiable provirus by qPCR  were amplified by nested PCR (nPCR) to confirm presence of provirus. The peak doubling time (T2) for proviral HTLV-1 was estimated from the sequential data as follows and a median result calculated:HTLV-1 proviral load was quantified by quantitative PCR (qPCR) as previously reported  with priy of tax  and two gag gene (5′ CTGGTGGAAATCGTAACTGGA-3′). Cycling conditions: 98 °C for 3 min, 35 cycles 98 °C for 10 s, 64 °C for 20 s, 72 °C for 20 s followed by 72 °C for 10 min. The PCR products were electrophoresed on a 2 % agarose gel, inspected for length, and sequenced by Sanger sequencing.To identify the genotype of the HTLV-1 provirus, we amplified a 523 bp fragment between the LTR (5′-CTCGCATCTCTCCTTCACG-3′) and the http://www.ncbi.nlm.nih.gov/nuccore/J02029.1) whilst the NCBI Blast database program was used to confirm specificity. Primer sequences were as follows: outer-pX-forward: 5′-ATGAGCCCCAAATATCCCCCGGGG-3′, outer-pX-reverse: 5′-TCGATCTGTAACGGCGCAGAAC-3′, nested-pX-forward: 5′-AGCCACCGGGAACCACCCAT-3′, nested-gag-reverse: 5′-GACAAAGGCCCGGTCTCGACCT-3′. Classical PCR: sample DNA isolated from a known number of cells was amplified in 50 μl reaction volumes containing 0.1 µM of each outer primer, 200 μM dNTPs, 2 mM MgCl2, 1× green GoTaq reaction buffer  and 1.25u GoTaq DNA polymerase (Promega). Cycling conditions on an MJ Research PTC-225  were: denaturation step 5 min at 94 °C, followed by 35 cycles of amplification consisting of 1 min at 95 °C, 30 s at 66 °C and 2 min at 72 °C, and a final elongation step of a further 5 min at 72 °C. For nested PCR, 1 µl of classical PCR product was transferred to 49 μl of reaction mix containing nested primers. Thermocycling conditions were: 94 °C for 5 min followed by 35 cycles consisting of 1 min at 95 °C, 30 s at 68 °C and 2 min at 72 °C, and a 5 min final elongation step at 72 °C. 10 µl of each reaction was separated on 2 % agarose gels containing 0.1 µg/ml ethidium bromide (Sigma) and visualised under UV light. LTR DNA circles were determined by serial dilution of purified sample DNA in water and amplification of quadruplicates at each dilution. LTR circle quantity was determined using Poisson’s distribution, where load = −logn Fox dilution, and Fo is the number of negative tests/the number of tests. MT2 cells served as positive controls, DNA from HTLV-1 negative donor blood mononuclear cells and water as negative controls. 2LTR DNA circle frequency was calculated as the absolute number of 2LTR DNA circles per infected cell.PCR primers  for detection of unintegrated HTLV-1 2LTR DNA circles were designed by alignment with the AKT strain of the complete HTLV-1 genome  . A PBMC The organs of the donor were offered for transplantation by the family of the donor, with written consent by the next-of-kin in accordance with the Human Tissues Act 2004 and the regulations of the Human Tissues Authority. In the United Kingdom this donation process and consent includes the testing of the donor for transplantation transmissible infections including HTLV-1.Each recipient gave written informed consent for the organ transplantation which is documented in the medical records. In the UK verbal consent alone is sufficient to obtain blood samples for clinical purposes and this is not separately documented. Although some new investigation techniques were applied in the clinical investigation of this unusual event these were not performed as part of a research study and therefore in accordance with the guidance of the relevant authority, which in England is the Health Research Authority, application to the National Research Ethics Service is not required. Furthermore, each recipient gave their consent for the HTLV investigations and separately and specifically each gave consent, documented in their medical records, for the publication of their case and the results of their investigations.


The prevalence of Metabolic Syndrome (MS) has been increasing worldwide. Although Uric Acid (UA) Levels are often increased in subjects with MS, it is still unclear whether uric acid plays a causal role for MS or is a marker. The purpose of this study was to examine the association between UA and the MS in Qazvin, Iran.529 men and 578 women aged 20 – 78 years attended in cross sectional study from September 2010 to April 2011 in Qazvin, Iran. The criteria proposed by new joint Interim societies (JIS) were applied for diagnosis of MS. Hyperuricemia was defined as UA ≥ 7 mg/dL in men and UA ≥ 6 mg/dL in women. Logistic regression analysis was performed to evaluate the relationship between UA quartiles and MS.The prevalence of MS was found to be 39.3%. Prevalence of hyperuricemia was 8.4% in males and 4.1% in females (P=0.004). Mean UA level was higher in males than in females (P<0.001). UA levels increased significantly with an increasing number of MS components in both genders. Prevalence of MS increased across UA quartiles in females; however the increasing trend began from second quartile in males. Using the lowest quartile of UA level as a reference, there were no significant association between UA quartile groups and MS.This study showed that UA levels are not an appropriate predictor of MS in Iranian population. More longitudinal studies are necessary to confirm the role of UA in MS occurrence. The metabolic syndrome (MS) refers to a cluster of risk factors, including abdominal obesity, high blood pressure, dyslipidemia and increased plasma glucose . MS is aLarge epidemiological studies have established the association of increased serum uric acid levels with MS and its individual components  was calculated as weight (kg) divided by the height (m) squared. Waist and hip circumference were measured to the nearest 0.1 cm using a flexible, non-elastic measuring tape without any pressure on the tissue. The waist circumference (WC) was measured halfway between the costal margin and the iliac crest at the end of normal expiration. The hip circumference (HC) was considered as the maximal circumference over the great femoral trochanters. Waist–to–hip ratio (WHR) was calculated as WC divided by HC. Blood pressure (BP) was measured three times – on a single occasion – in a seated position using a mercury sphygmomanometer after a 15 min rest. There was an at least 2 minute interval between last two measurements and their average value was registered as subject’s BP. If an abnormal value was obtained, then another measurement was taken after a 30 minute rest.A venous blood sample of subject was taken after a 12 – 14 hour overnight fast. All the samples were analyzed at the same laboratory on the day of blood collection. Blood levels of glucose, insulin, total cholesterol (Chol), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), triglycerides (TGs), creatinin (Cr) and uric acid (UA) were measured in all subjects.A Hitachi 704 auto-analyzer with GOD-PAP method and reagent  was used to measure fasting blood glucose (FBS); Mean intra- and interassay coefficients of variation (CVs) were 1.28 and 0.84, respectively. An oral glucose tolerance test (OGTT) was performed on every subjects who had never been diagnosed with diabetes. Impaired glucose tolerance was defined as a glucose level greater than 140 mg/dL (7.8 mmol/L) but less than 200 mg/dL (11 mmol/L) at two hours. Insulin levels were measured by ELISA using reagent . A within-run precision CV was 4.9 and total precision CV was 4.9. A Hitachi 704 auto-analyzer with CHOD-PAP and reagent (Parsazmun Company) was used to measure cholesterol; Mean intra- and interassay CVs were 0.61 and 1.22, respectively. A Hitachi 704 auto-analyzer with Immunoturbidimetric and reagent (Parsazmun Company) was used to measure HDL-C and LDL-C; Mean intra- and interassay CVs for HDL-C were 0.73 and 1.8, respectively; for LDL-C they were 0.63 and 1.29, respectively. A Hitachi 704 auto-analyzer with GPO-PAP and reagent (Parsazmun Company) was used to measure TGs; Mean intra- and interassay CVs were 1.82 and 1.04, respectively. A Hitachi 704 auto-analyzer with Uricase and reagent (Parsazmun Company) was used to measure UA; Mean intra- and interassay CVs were 1.18 and 1.13, respectively.Insulin resistance (IR) was estimated by the homeostatic model assessment (HOMA-IR), as fasting serum insulin (μIU/ml) × fasting plasma glucose (mmol/L) /22.5 .Kolmogorov Smirnov test was used to examine the normality of variables of interest. Data were recorded as mean plus or minus standard deviation (SD) for normally distributed variables or as median values (minimum – maximum) for non–normally distributed variables. Categorical variables were analyzed by chi square test, T-test was used for analysis of continuous variables and non-normally distributed variables were compared by Mann Whitney U test. The relationship between UA level and other variables including MS components were assessed by Pearson’s correlation coefficients. Logistic regression analysis was performed to evaluate the relationship between UA quartiles and MS. Various variables were considered as the potential confounding factors in five models. P-values less than 0.05 were considered statistically significant.The study was performed for a total of 529 men and 578 women aged 20- 78 years (40.08 ± 10.33). UA levels increased significantly with an increasing number of MS components in both genders . Post HoUA levels of subjects with and without MS and every component of MS has been shown in Subjects with hyperuricemia had higher prevalence of every component of MS with the exception of low HDL-C and a 3.23-fold higher risk of MS in comparison with normouricemic subjects. Among the MS components, hypertriglyceridemia demonstrated the strongest association with hyperuricemia .In both genders, UA levels were positively and significantly correlated with WC, BP and TGs. UA levels were significantly and negatively correlated with HDL-C in females. UA continued to have a significant correlation with WC and TGs level in both genders and with HDL-C in females after adjusting for age, Cr and cholesterol. Otherwise, there was significant and negative correlation between UA levels and FBS in males after this adjustment.The prevalence of MS according to UA quartiles has been shown in Using the lowest quartile of UA level as a reference, crude and adjusted multivariate odd’s ratios (ORs) were calculated according to various models. Association between UA level quartiles and MS using logistic regression analysis has been shown in Elevated serum UA level is associated with cardiovascular risk factors including hypertriglyceridemia, hypertension, obesity, and hyperglycemia. Presence of these factors together in the same patient is known as MS . UA leveThe prevalence of hyperuricemia in the present study was far lower than in many related studies include studies on Iranian population . Higher The Present study showed that UA levels were significantly higher in subjects who had increased number of MS components. This relationship was also significant in both genders. These findings have been reported in other studies . On the other hand it has been proposed that UA may be a causative factor for MS. Hyperinsulinemia increases tubular sodium resorption that results in impaired UA excretion. So, normoglycemic subjects may have higher UA level in the presence of insulin resistance . So, it UA levels were positively correlated with WC and TGs level in both genders and were negatively correlated with HDL-C in females in the present study. Unexpectedly negative correlation between UA levels and FBS was found in males that had been also reported by Lu et al formerly (We found positive correlation between UA levels and MS and its components including TGs level, BP and WC in studied subjects. Independent correlation of TGs with UA levels has been reported by previous studies; but its underlying biological mechanism has not been elucidated. This association may be due to both environmental and genetic factors (After adjustment for different confounders, there was no significant correlation between UA levels and BP. As WC was a main determinant of UA levels in present study, it may be due to the effect of obesity on SBP and DBP. Negative correlation between UA levels and HDL-C is compatible with studies in Hangzhou and Taiwan .An increase in the prevalence of MS with increasing UA levels was found in the present study that is consistent with previous studies performed on various ethnic groups. These studies have not explained that whether the relationship between UA and MS is independent or not . PositivThe multivariate unadjusted ORs between UA quartiles and MS were less than 1 in the present study which was increased after adjustment for different variables in the models. This is in contrast with findings of Lim et al and Meshkani et al studies in which ORs more than 1 decreased after adjustment (Meshkani, Zargari, & Larijani, 2010; When we added Cr, cholesterol levels and WC in adjustment model, the ORs of all quartiles was increased; but the relationship was not statistically significant with regards to confidence interval and P-values. Same results have been reported by the above two mentioned studies. Such results imply that the association between UA levels and MS is not independent, but is affected by various confounders such as physical activity and smoking. In Onat et al study, the ORs of the upper UA tertile were significantly decreased in both genders; but lost significance in males after adjustment for WC (The present study had some limitations include the cross sectional basis of its design and the number of studied subjects. Some confounders include physical activity; the use of diuretics and a diet habitually high in purines content were not considered. Other unknown confounders may also affect the results.UA level is not a part of JIS criteria or other similar criteria for definition of MS. The association between UA levels and MS is not independent, but is affected by various confounders such as Cr and cholesterol levels, WC, physical activity and smoking. Results of the present study showed that UA levels are not an appropriate predictor of MS in Iranian population. Relatively low prevalence of hyperuricemia, low purine content of Iranian diet and high prevalence of MS may explain these results. More longitudinal studies are necessary to confirm the role of UA in MS occurrence.


HTLV prevalence is poorly mapped in several areas, in particular in Africa. Global prevalence is commonly cited as being 10-20 million but more recently this has been contested as an overestimation. Additional epidemiological studies would further our understanding of the HTLV burden. Similarly, HCV is endemic in certain locations (Egypt) whilst data for other areas of Africa are virtually absent. We investigated HTLV/HCV prevalence in serum samples from 418 paired mother and child samples of healthy mothers of children attending Malawian hospitals. Latest generation ELISAs were used; consistently ELISA reactive mothers’ sera were further tested by immunoblot and only deemed sero-positive if they were reactive in this assay. Serum from the children of ELISA positive/borderline mothers were subsequently screened. Sero-prevalence in the healthy women was as follows, HTLV-1: 0.72% (n=3), HTLV-2: 1.9% (n=8), HTLV indeterminate: 3.8% (n=16) and HCV: 0.48% (n=2). DNA extracted from whole blood could only resolve one of the high percentage of intermediate positive results, most likely because of a low proviral load. One child in each category: HTLV-1, HTLV-2, dual-infected and HTLV indeterminate was identified indicating low (11.54%) mother to child transmission (MTCT) whereas one of the two HCV positive mothers had a HCV positive child, suggestive of high HCV MTCT transmission rate. We conducted a systematic review to compare this original data with previous reports: Malawi had similar, low, levels of HTLV-1 and HCV but surprisingly high HTLV-2. Prevalence maps for HTLV and HCV for this demographic in the African continent and the meta-analysis will be presented.


The association between maternal serum concentration of betamethasone given for fetal lung maturity and perinatal outcome has not been investigated. This may be due to an absence of a reliable method for measuring serum betamethasone concentrations. We aimed in the current study to assess the feasibility of a specific ELISA kit to measure the concentrations of betamethasone in maternal serum and to examine the trend of sequential measurements after a course of betamethasone for fetal lung maturity.st dose. Betamethasone concentration in samples was determined using Corticosteroid ELISA kit. The Friedman test was used to test whether there were significant differences between the measurements.Pregnant women at risk for preterm birth who received betamethasone between 24 and 34 weeks of gestation were prospectively included. Serum concentrations were determined before administering betamethasone (baseline), and 36 hours, 48 hours, 72 hours, and 5 to 7 days after the 1st dose and close to baseline at the 5th measurement performed after 5 to 7 days (p < 0.05). Serum concentration varied at each time point between the five women but similar trend was observed.Five singleton pregnancies were included. Using the ELISA kit, betamethasone concentration in maternal serum samples was obtained for all women. Among the five measurements performed, the concentration was highest at 36 hours after the 1Betamethasone concentration is measurable in the serum of pregnant women with this ELISA kit. Preterm birth is a major contributor to perinatal mortality and morbidity and affects approximately 7 to 12% of births in developed countries .Liggins’ 1969 study with sheep that found improved survival for preterm lambs delivered after corticosteroid exposure , paved tThe currently accepted dosage of betamethasone was determined by Liggins and Howie in the 1970s; since that time, pregnant women at risk for preterm birth have been administered a fixed dosage of betamethasone, regardless of gestational age, maternal body mass index, and the presence of a singleton or multiple gestation . AlthougTo the best of our knowledge, the relationship between betamethasone concentration and perinatal outcome has not been reported. In part, this may be due to an absence of a reliable method for measuring serum betamethasone concentrations. In view of that, we aimed in this study to assess the ability of a specific ELISA kit to measure the concentrations of betamethasone in maternal serum and to examine the trend of sequential measurements in maternal serum after a complete course of betamethasone for lung maturity.This prospective study was held at a university teaching medical center between July 2012 and April 2014. Pregnant women between 24 weeks 0 days and 33 weeks 6 days of gestation, had singleton gestation, and received a complete course of betamethasone due to threatened preterm birth were included. A complete course of betamethasone (consisting of 50% betamethasone phosphate and 50% betamethasone acetate) involved two 12 mg intramuscular doses 24 hours apart. Women who received an incomplete course of betamethasone because of failure to delay delivery or who received corticosteroids for other reasons during pregnancy, had multiple gestations, or fetal malformations diagnosed in the antepartum period were excluded from the study.In order to examine whether betamethasone levels are measurable and in order to examine the dynamics of betamethasone concentrations against time from treatment, blood samples were drawn from each participating woman at five time points: immediately before administering the betamethasone (baseline); at 36 hours after the first dose; at 48 hours after the first dose; at 72 hours after the first dose, and 5 to 7 days after the first dose, given that a preterm birth did not occur earlier. These consecutive measurements were chosen in order to examine whether the trend of concentrations acted comparably to the anticipated clinical effect of betamethasone, which is best achieved within 7 days from the initial dose .In order to examine whether half-life (t1/2) of betamethasone concentration varies during different periods after betamethasone administration and between the participating women, t1/2 of betamethasone was calculated between the second and third measurements (36 to 48 hours after the first injection) and between the third and fourth measurements (48 to 72 hours after the first injection) for all women. Calculation was performed using the formula t1/2 = t * ln(2)/ln(N0/Nt) where, t = time between measurements, ln = natural logarithm, N0 = concentration at the beginning, and Nt = concentration at the end of the time between the two measurements.Serum samples were kept at -70°C until processing. Betamethasone concentration in samples was determined using the Corticosteroid ELISA kit  according to the manufacturer’s instructions, processed in a Triturus apparatus . This kit, originally designed for urine, tissue, and serum samples in the veterinary field, was evaluated in our laboratory for use with human samples. Evaluation included precision, repeatability, linearity, and analytical sensitivity (lower limit of detection). Those parameters were analyzed by spiking betamethasone (provided with the kit) and the same generic betamethasone that was administered to women into a pool of human sera. According to linearity range obtained in the evaluation, study samples were diluted accordingly in order to be measured within this range.The present study was approved by the ethics committee of Emek Medical Center in compliance with the Helsinki declaration. Signed consent was obtained from each of the participating women before receiving the first betamethasone injection.p < 0.05. For continuous variables, the Student t-test was implemented. SAS 9.2 was used to perform the statistical analyses and the graphical presentation .The mean and standard deviation of the betamethasone levels from all samples were calculated. The Friedman test was used to determine whether there were significant differences between betamethasone concentrations. Significance level was set at A total of eight women were approached and consented. Three women delivered before receiving a complete course of betamethasone and for that reason they were excluded. Overall, five women (A to E) received a complete course of betamethasone and were included in the study. Demographic and obstetric parameters are presented in Table th measurement performed 5 to 7 days after the first dose of betamethasone. The trend between measurements was significant (p < 0.05). Although the trend of sequential measurements was similar for the five women, there were obvious variations at each time point .Calculated half-life of betamethasone differed according to the time elapsed from its administration Table . Mean haThe evaluation of betamethasone measurement kit performance demonstrated a high precision for spiked betamethasone in the human sera pool: average coefficient of variation of 3.5 and 6.1% for kit and generic betamethasone, respectively, for expected values between 0.1 and 50 ng/mL. The kit demonstrated a good linearity (average bias = ±4.0%) for the range between 0.1 and 50 ng/mL. Analytical sensitivity (lower limit of detection) was 0.05 ng/mL.th measurement at 5 to 7 days. The trend of sequential measurements performed similarly between all women; however the concentration varied at each time point between the five women. The near to baseline concentration at 5 to 7 days is similar to the described perinatal clinical effect that is best achieved within 7 days after drug administration [In this study we were able to measure betamethasone concentrations in the serum of pregnant women with the use of corticosteroid ELISA kit. Among the measurements performed for each woman, the concentration was highest at 36 hours after the first dose of betamethasone followed by a gradual decrease to a level close to the baseline at the 5stration .The National Institutes of Health and the American College of Obstetricians and Gynecologists recommend routine glucocorticoid administration to women at risk for preterm birth between 24 and 34 weeks gestation to accelerate fetal lung maturation and to decrease the incidence of other neonatal morbidity and mortality related to prematurity , 12. WheAlthough betamethasone treatment reduces mortality and morbidity in general, its effect on a particular woman is usually unpredictable. The current dosage of betamethasone was determined by Liggins and Howie during the 1970s, and since then a fixed dosage is administered to all pregnant women regardless of gestational age, maternal body mass index, and whether a singleton or multiple gestation is present . AdditioLoehle et al. reported that routine therapy of two 12 mg doses of betamethasone phosphate alone given intramuscularly to the average weight mother 24 h apart is supramaximal for fetal lung maturation. Their data showed that half the normal clinical dose, adjusted for weight, may be functionally adequate . On the The half-lives of betamethasone concentration found in the current study differ from the fixed 9.0 ± 2.7 hours reported by Ballabh et al. . This maThe results of the current study do not provide evidence as to the precise dosage of betamethasone that needs to be administered; the minimum betamethasone dosage that would be clinically effective is yet to be determined. Basic pharmacologic principles suggest that the drug concentration in maternal serum may determine the fetal response and perinatal outcome and, as such, the optimal treatment regimen. Given that, the results of the current study may pave the way for future studies that could determine the optimum concentration and, accordingly, the dosage of betamethasone by investigating the minimum concentration that would be clinically effective to positively affect perinatal outcome.


Human T-lymphotropic virus type-1 (HTLV-1) persists within hosts via de novo infection (infectious spread) and infected cell proliferation (mitotic spread), creating a population structure of multiple clones . However, the relative contributions of these mechanisms are unknown, and will determine the efficacy of antiretroviral therapy. The prevailing view is that infectious spread does not contribute to HTLV-1 proviral load after early infection. However, this notion is based on low-throughput and imprecise data on clonal integration site abundance and diversity. We recently used our high-throughput protocol that maps and quantifies the abundance of HTLV-1 integration sites to estimate a diversity of between 10^4 and 10^5 HTLV-1 T cell clones in the body of an asymptomatic carrier or patient with HAM/TSP. Since infectious spread establishes new clones, high clonal diversity implies substantial infectious spread. HTLV-1 clonal abundance varies within individual hosts by several orders of magnitude. Therefore quantifying within-host HTLV-1 dynamics requires mathematical modelling at multiple scales, and stochastic processes are necessary to describe the behaviour of small clones. Here we apply a hybrid model of deterministic and stochastic processes to time-series patient data, to estimate the relative contributions of infectious spread and mitotic spread. We find, contrary to previous belief that infectious spread persists during chronic infection, even after the proviral load has reached its set point. The risk of HTLV-1-associated malignant and inflammatory disease is strongly correlated with the proviral load: the load in turn is correlated with the total number of HTLV-1-infected clones, but not with the degree of oligoclonal proliferation. Our results therefore suggest that attempts to suppress de novo infection may reduce the risk of malignant transformation.