We induced glutathionylation in vitro by treatment of isolated myofibrils and detergent extracted fiber bundles (skinned fibers) with oxidized glutathione (GSSG). Immuno-blotting results revealed increased glutathionylation with GSSG treatment of a protein band around 140 kDa. Using tandem mass spectrometry, we identified the 140 kDa band as cMyBP-C and determined the sites of glutathionylation to be at cysteines 655, 479, and 627. Determination of the relation between Ca2+-activation of myofibrillar acto-myosin ATPase rate demonstrated an increased Ca2+-sensitivity induced by the S-glutathionylation. Force generating skinned fiber bundles also showed an increase in Ca-sensitivity when treated with oxidized glutathione, which was reversed with the reducing agent, dithiothreitol (DTT). Our data demonstrate that a specific and direct effect of S-glutathionylation of myosin binding protein C is a significant increase in myofilament Ca2+-sensitivity. Our data also provide new insights into the functional significance of oxidative modification of myosin binding protein C and the potential role of domains not previously considered to be functionally significant as controllers of myofilament Ca2+-responsiveness and dynamics.Our previous studies demonstrated a relation between glutathionylation of cardiac myosin binding protein C (cMyBP-C) and diastolic dysfunction in a hypertensive mouse model stressed by treatment with salt, deoxycorticosterone acetate, and unilateral nephrectomy. Although these results strongly indicated an important role for S-glutathionylation of myosin binding protein C as a modifier of myofilament function, indirect effects of other post-translational modifications may have occurred. Moreover, we did not determine the sites of thiol modification by glutathionylation. To address these issues, we developed an  Free Ca2+ concentration was calculated using WEBMAXC STANDARD. We determined myofibrillar ATPase activity at 30°C by starting the reaction with 1 mM ATP and stopping the progress by addition of trichloroacetic acid every 3 min for 15 min, during which Pi generation, as determined with a malachite green based assay, was linear  and Hill n values.Four month old female FVBN mice were deeply anesthetized with 60 mg/kg pentobarbital. The heart was quickly excised and rinsed in cold 0.9% sodium chloride. All methods were approved by the University of Illinois at Chicago Animal Care and Use Committee. We isolated myofibrillar fractions from ~50 mg wet weight of left ventricular tissue using a modification of procedures described by Solaro et al.  and Layl2+ relationship were carried out on fiber bundles from left ventricular papillary muscle of adult mice essentially as previously described , leupeptin (1 ug/ml) and phenylmethylsulphonyl fluoride . Fiber bundles (150–200 μm in width and ~4–5 mM long) were dissected from papillary muscles. Membranes were extracted from the fiber bundles by immersing them for 30 min in HR buffer containing 1% Triton X-100. We mounted the fiber bundles between a force transducer and micromanipulator, and, after an initial contraction to maximum force and return to relaxation, we set the sarcomere length at 2.2 μm using laser diffraction patterns. A, The fibers were then exposed to solutions of incrementally increasing Ca2+ concentrations ranging from 10−7 to 10−4.5 M and force was recorded to determine the force-pCa relations. Fibers were then incubated in 5 mM GSSG in A-70 as prepared above for 10 min, and then subjected to the varying Ca2+ concentrations. This process was repeated using 10 mM dithiothreitol (DTT) solubilized in A-70 for 10 min. No changes in force or Ca-sensitivity were observed when we treated control skinned fibers (no BSSG) with DTT. Tension was calculated by dividing the force by the cross-sectional area, as described previously  according to manufacturer's protocol. The band around 140 kDa (MyBP-C) was cut from the gel and subjected to in-gel digest with Trypsin Gold (Promega). Reduction and alkylation steps were omitted to preserve the S-glutathionylation of the proteins. Pooled digestion extracts were concentrated via Speed Vac to less than 20 μl and brought up to 40 μl with mobile phase solution (5% ACN and 0.1% formic acid). Peptides were filtered with 0.22 μm PVDF Millipore Ultrafree-MC spin filter and 35 μl of sample was analyzed with Thermo Finnigan LTQ hybrid linear ion trap—Fourier Transform ICR mass spectrometer coupled to Dionex U3000 nano LC. Dionex acclaim PepMap100 C18 trapping column  was used to concentrate the samples at flow rate of 50 μl/min and Agilent Zorbax 300SB-C18 Nanoflow column  was used for separation at flow rate of 0.250 μl/min. Peptides were eluted with a linear gradient of 5–45% solution B  through New Objective uncoated SilicaTips™ . Peptides were ionized via electrospray ionization with LTQ source voltage set to 1.0 kV and capillary temperature set to 200°C. Mass spectra were obtained in positive ion mode over m/z range of 400–1800 at a resolution of 50,000 and top ten most intense ions were selected for tandem MS. MS/MS were obtained in collision induced dissociation mode with minimum signal required 5000, isolation width 3, and normalized collision energy 35.Mass spectrometry data were collected with Xcalibur 2.0.7 software as.Raw file, which were converted to mzXML and MGF files using MassMatrix MS Data File Conversion tool  was 6.50 ± 0.07 (n = 4) compared to a pCa50 of 6.17 ± 0.09 (n = 4) for controls. With 5 mM GSSG the pCa50 was 6.60 ± 0.03 (n = 4) compared to controls with pCa50 of 6.02 ± 0.07 (n = 4). The Hill coefficients, minimum, and maximum ATPase rates did not change significantly between the control myofibrils and myofibrils treated with either 1 or 5 mM GSSG.In order to determine direct effects of glutathionylation on myofilament response to calcium, we compared ATPase activities of isolated myofibrillar preparations before and after treatment with 1 and 5 mM GSSG Figure  or 5 mM 2+, sensitivity of ATPase rate for the GSSG treated myofilaments, we analyzed the samples by Western blot analysis. Figure To determine the mechanism for the higher Ca2+ sensitivity was significantly higher after GSSG incubation as indicated by pCa50 of 6.15 ± 0.01 (n = 4) compared to controls with pCa50 of 6.09 ± 0.01 (n = 4). This increase in pCa50 with direct glutathionylation is similar the increase in pCa50 we reported when comparing myofilaments sham mice and DOCA/salt mice [Figure 4A in Jeong et al. (2+ sensitivity associated with GSSG treatment was reversed after incubation in DTT as indicated by pCa50 of 6.03 ± 0.02 (n = 4). Cooperativity of activation, as measured by the Hill slope tended to decrease after GSSG incubation and return to control levels after DTT incubation, but none of the changes were statistically significant. As with the myofibrillar preparations, Western blots of the skinned fiber preparations revealed glutathionylation of MyBP-C  (Prochniewicz et al., 2+sensitivity.Although it is likely that other proteins in cardiac sarcomeres are S-glutathionylated, based on the present results and our previous data, the impact of S-glutathionylation of MyBP-C appears to be of relatively high significance in controlling cardiac relaxation and as an important mechanism for the diastolic dysfunction associated with hypertension induced oxidative stress. There have been reports that reversible glutathionylation of actin at Cys479, Cys627, and Cys655. The modification in the C5 domain may be of particular interest inasmuch as this domain contains a cardiac specific region. Some evidence points to a functional significance of these C-terminal domains of cMyBP-C in the heart. Interactions of this region with titin and light meromyosin have been documented but not extensively analyzed in terms of functional significance (Yang et al., Our data indicate high susceptibility of cMyBP-C for modification by glutathionylation at specific cysteine residues, which may be exposed in the in the three dimensional structure for ease of access by GSSG. Glutathionylation has also been correlated with relatively low pKa values for susceptible Cys residues (Pastore and Piemonte, In conclusion our data emphasize and support our earlier data indicating the potential for redox related post-translational modification of MyBP-C as a significant factor controlling cardiac function. Our results also emphasize the need for better understanding of the role of the C-terminal regions of MyBP-C that contain thiols highly reactive with GSSG. The specificity of the correlation of S-glutathionylation with myofilament function also indicates that effects of oxidative stress need to be considered in the context of the particular circumstances generating ROS. It will be important in further studies to determine interactions among these redox related post-translational modifications and phosphorylation. Whether the existence of a S-glutathionylated form has any relevance in a variety of cardiac disorders needs to be further explored. Our findings may also relate to the use of serum levels of MyBP-C as a biomarker for acute myocardial function (Jacquet et al., Bindiya G. Patel performed gel analysis, the ATPase measurements, and mass spectrometry. Tanganyika Wilder performed the force measurements and gel analysis. Bindiya G. Patel and Tanganyika Wilder also wrote drafts of the manuscript. R. John Solaro designed the experiments and supervised all aspects of the study and wrote the final draft with input from Bindiya G. Patel and Tanganyika Wilder.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.


Thymic Stromal Lymphopoietin (TSLP) is a potent immunomodulatory cytokine involved in Th2-mediated immune responses and homeostatic T-cell expansion. Reduced TSLP expression by intestinal epithelial cells was recently shown to lead to reduced Th2 responses and development of Th1-mediated experimental colitis. Additionally, TSLP is described as a proinflammatory factor in autoimmune diseases. TSLP expression was studied in salivary glands of primary Sjögren’s syndrome (pSS) patients as compared to non-SS Sicca (nSS) patients and the relationship to local- and systemic disease parameters was examined.Tissue sections of minor salivary glands from 38 pSS and 18 nSS patients were stained with a monoclonal antibody (mAb) against human TSLP or an isotype control. In addition, sections were stained with mAbs against CD3, CD19 and epithelial cell marker cytokeratin high molecular weight (CK HMW) or stained with alcian blue to detect mucus production. The number of TSLP-expressing cells was quantified and expression was correlated to local- and systemic disease parameters.2 was significantly decreased in pSS patients as compared to nSS patients and correlated negatively to Lymphocyte Focus Score (LFS), ESR, serum IgG levels and positively to the percentage of local IgA producing plasma cells. In pSS patients, TSLP was expressed outside of lymphocytic infiltrates at sections that also encompassed high numbers of intact, functional acinar cells as analyzed with CK HMW and Alcian Blue staining.TSLP was almost exclusively expressed by acinar cells in both pSS and nSS patients. The number of TSLP-expressing cells per mmTSLP expression is reduced in pSS patients, associated with local- and systemic inflammatory markers.Considering the described role of TSLP in promoting Th2 responses at mucosal sites, we hypothesize that TSLP is constitutively expressed in salivary glands and promotes a protective Th2 milieu, whereas loss of TSLP expression may contribute to Th1/Th17 associated immunopathology in pSS.


Gene expression profile (transcriptome) and glycan profile (glycome) of HTLV-1-infected CD4+T cells may reflect the pathologic cellular mechanism and intercellular recognition, respectively in HAM/TSP though these profiles are still not obtained. To identify responsible cellular genes and relevant glycans of HAM/TSP, we performed experiments with microarray on RNAs and and lectin array on proteins extracted from CD4+Tcells from each four subjects of three goups including HAM/TSP, asymptomatic carriers (AC), and HTLV-1 negative controls (NC). In transcriptome analysis, transcripts of 177 genes were found up-regulated only in HAM/TSP, and those may possibly be causative or resultant genes. In glycome analysis with lectin array carrying 45 species of lectins, standardized signals of UDA  and STL  which recognize N-glycan (GlcNAc)n were significantly high in samples from HTLV-1 infected groups (HAM/TSP and AC) compared with NC. Interestingly, UDA has been recently reported to inhibit cell-to-cell transmission of HTLV-1 in vitro. These genes and glycans may play roles in the pathogenesis of HAM/TSP.


HTLV-1 is capable of transforming T lymphocytes in infected patients resulting in adult T cell leukemia/lymphoma whereas HTLV-2 is not clearly associated with lymphoproliferative diseases. Numerous studies have provided accumulating evidence on the involvement of the viral transactivators Tax-1 versus Tax-2 in T cell transformation. Tax-1 is a potent transcriptional activator of both viral and cellular genes. Tax-1 post-translational modifications and specifically ubiquitylation and SUMOylation have been implicated in nuclear factor-kappaB (NF-κB) activation and may contribute to its transformation capacity. Although Tax-2 has similar protein structure compared to Tax-1, the two proteins display differences both in their protein–protein interaction and activation of signal transduction pathways. Recent studies on Tax-2 have suggested ubiquitylation and SUMOylation independent mechanisms of NF-κB activation. In this present review, structural and functional differences between Tax-1 and Tax-2 will be summarized. Specifically, we will address their subcellular localization, nuclear trafficking and their effect on cellular regulatory proteins. A special attention will be given to Tax-1/Tax-2 post-translational modification such as ubiquitylation, SUMOylation, phosphorylation, acetylation, NF-κB activation, and protein–protein interactions involved in oncogenecity both  Although currently there are four known types of HTLV retroviruses , 2009, Hormation . HTLV-2,ormation . In contormation . HTLV-2 ormation , native ormation , and amoormation .Both Tax-1 and Tax-2 are required for HTLV-1 and HTLV-2 viral replication and they play an important role in proviral transcription . In addiFigure 1A), but have distinctive differences both at the structural and functional levels .Tax-1 and Tax-2 share overall sequence homology . The CREB domain is required for activation of the viral promoter . Furthermore, Tax-2 has an additional cytoplasmic localization domain about 10 amino acids long, situated at amino acid position 89–113 which has been shown to be responsible for its divergent localization compared to Tax-1 -activating domain and a zinc finger domain . Importantly, Tax-2 lacks these two LZR regions. Both Tax-1 and Tax-2 have nuclear export signal (NES) located at amino acid position 189–202 . Furthermore, Tax-1 and Tax-2 have at the C-terminal region CREB/activating transcription factor (ATF)-activating domain, essential for transactivation of the CREB/ATF and for NF-κB/Rel signaling pathways .The central region of Tax-1 includes two leucine zipper-like regions (LZR), which are known to be essential for protein dimerization and DNA interaction . The firin vitro with a number of proteins of the CREB/ATF family of transcription factors: CREB, CREM (cyclic AMP responsive element modulator), ATF1, ATF2, ATF3, ATF4 , and XBP1 . Indeed, PDZ domain-containing proteins play a key role in recruiting and organizing the appropriate proteins to sites of cellular signaling, as well as polar sites of cell–cell communication (Drosophila melanogaster disc large tumor suppressor protein hDLG (homolog of Drosophila disc large), which regulates cellular proliferation and cell cycle phase transition  SMYD3 which affects its nucleo-cytoplasmic shuttling and regulates NF-κB activation . InteracTax-1 interacts with various components of the cell signaling system which control cell transformation, proliferation, intracellular protein distribution, cell migration, and virological synapses . More th473 which in turn activates a broad range of regulatory proteins and transcription factors such as AP1  and its downstream kinase AKT/PKB (protein kinase B) are activated in T cells by many cytokines including interleukin 2 (IL-2), and provide cell survival and growth signals . PI3K ach as AP1 . In bothh as AP1 . The PI3h as AP1 . Other sh as AP1 . Tax-1 hh as AP1 . Conversagosomes .Mitogen-activated protein kinases (MAPKs) are serine/threonine-specific protein kinases that respond to external mitogen stimuli such as growth factors, cytokines or physical stress. MAPK signaling involves a sequential phosphorylation cascade of MAP kinase kinase kinase (MAP3K). There are at least five distinct MAPK subgroups: the extracellular signal-regulated kinases protein homologs 1 and 2 (ERK1/2), the big MAPK-1 (BMK-1) also referred to as ERK5, the stress-activated protein kinases-1 (SAPK-1) better known as the c-Jun N-terminal kinase homologs 1, 2, and 3 (JNK1/2/3), the (SAPK-2) homologs (p38α/β/d) and finally ERK6 also known as p38γ . Tax-1 bTransforming growth factor β (TGFβ) inhibits T cell growth in mid-G1 but can also promote tumorigenesis . TGFβ biAdult T cell leukemia/lymphoma cells produce high levels of TGFβ in the sera of HTLV-1-infected patients due to constitutive activation of AP-1 in the PI3K/AKT pathway . Tax-1 bThe guanine nucleotide-binding proteins GTPases (G proteins) are molecular switches that cycle between active (GTP-bound) and inactive (GDP-bound) states. Tax-1 forms complexes with several members of the small GTPase Rho family G proteins such as RhoA, Rac, Gap1m, and Cdc42 . Rho GTPFigure 1). This PDZ domain might contribute to Tax-1 binding to proteins involved in microtubule and cytoskeleton organization, which in turn may play an important role in pathogenicity and transformation capacity . Tax-1 and Tax-2 modulate CREB and ATF function . Tax-1/TBoth Tax-1 and Tax-2 interact with a series of CREB/ATF factors and modulate expression of viral and cellular genes through CRE elements. However, the specific binding of each CREB/ATF member still needs to be studied, although some in vitro analysis suggest Tax-1 interaction with a number of proteins of the CREB/ATF family of transcription factors: CREB, CREM, ATF1, ATF2, ATF3, ATF4 , and XBP1 .in vitro studies in different cell types have shown that Tax-1 represses p53 activity through different mechanisms including NF-κB activation and/or the CREB pathway , c-Rel, RelB, p50/p105, and p52/p100. The precursor proteins p105 and p100 are processed proteolytically to the mature p50 and p52 forms, respectively . Tax-1 therefore, connects activated kinases to the IKK complex and forces the phosphorylation of IKK-α and IKK-β leading to IKK activation, which results in phosphorylation, ubiquitylation, and proteasome-mediated degradation of IκBα and IκBβ . In fact, silencing of MEKK1 and TAK1 does not impair Tax-1-induced NF-κB activation . Tax-1 therefore, leads to IκB degradation at multiple levels, thereby allowing nuclear translocation of NF-κB independently of external stimuli. In the non-canonical pathway, Tax-1 interacts with IKK-γ (NEMO) and p100, induces p100 processing and nuclear translocation of the p52/RelB dimer . It therefore appears that IKK-γ is an important Tax-1-binding partner for activation of both pathways . The LZR at amino acid 225–232 of Tax-1, which is missing in Tax-2, is responsible for p100 processing and p52 nuclear translocation . Adjacent serine residues at positions 300 and 301 in the carboxy-terminus of Tax represent the major sites for phosphorylation. Indeed, phosphorylation of at least one of these serine residues is required for Tax localization in nuclear bodies and for Tax-mediated activation of gene expression via both the ATF/CREB and NF-κB pathways . Tax-1 acetylation in turn participates in NF-κB activation . Five of these residues located within Tax-1 C-terminal region were found to be the major targets ubiquitylation , whereas SUMOylation takes place on Lys-280 (K7) and Lys284 (K8)  .Tax-1 is indeed differentially ubiquitylated by either K-48 ubiquitin chains leading to Tax degradation by the proteasome or by K-63 ubiquitin chains that mediates IKK recruitment to the centrosome and IKK activation . On the The involvement of Tax-2 SUMOylation and ubiquitylation in NF-κB activation remains controversial. in vitro Tax-1 inducible cell lines  messenger and protein .To date, vast amount of knowledge has been produced regarding the HTLV-1 Tax-1 oncoprotein. Many studies have provided some insights on Tax-1 transcriptional regulation, subcellular localization and post-translational modifications. However, less is known about HTLV-2 Tax-2 although many aspects of its activity and regulation is now being studied. That HTLV-2 is defective in promoting certain steps of leukemogenesis, may indeed serve as a useful comparative tool for understanding the pathogenicity of HTLV-1.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.


Myelodysplastic syndromes (MDS) are often accompanied by autoimmune phenomena. The underlying mechanisms for these associations remain uncertain, although T cell activation seems to be important. Human T-lymphotropic virus (HTLV-1) has been detected in patients with myelodysplastic syndromes, mostly in regions of the world which are endemic for the virus, and where association of HTLV-1 with rheumatological manifestation is not rare. We present here the case of a 58 year old man who presented with cytopenias, leukocytoclastic vasculitis of the skin and glomerulopathy, and was diagnosed as MDS (refractory anemia with excess blasts - RAEB 1). The patient also tested positive for HTLV-1 by PCR. After 8 monthly cycles of 5-azacytidine he achieved a complete hematologic remission. Following treatment, a second PCR for HTLV-1 was carried out and found to be negative. This is the first report in the literature of a HTLV-1-positive MDS with severe autoimmune manifestations, which was treated with the hypomethylating factor 5-azacitidine, achieving cytogenetic remission with concomitant resolution of the autoimmune manifestations, as well as HTLV-1-PCR negativity. HTLV-1-PCR negativity may be due to either immune mediated clearance of the virus, or a potential antiretroviral effect of 5-azacytidine. 5-azacytidine is known for its antiretroviral effects, although there is no proof of its activity against HTLV-1 infection in vivo. The association of myelodysplasia with autoimmune phenomena is well established for more than 20 years, as evidenced by several case reports and small patient series. HTLV-1, the first described human retrovirus, and cause of adult T cell leukemia/lymphoma (ATLL) has been associated to myelodysplastic syndromes. In areas endemic for the virus, serologic positivity for HTLV-1 is not very rare in patients with myelodysplastic syndromes . These cA 58 year old man was admitted to our clinic due to a 6-month history of night sweats, anorexia, and weight loss. 6 months ago he had an influenza-like illness and 4 months before admission, he developed ptosis of his left eyelid, with no other neurologic signs and symptoms and a negative brain MRI and carotid artery triplex ultrasound. The symptom resolved without any neurologic sequelae. One month before admission, he developed erythema nodosum on his left leg. His past medical history was unremarkable.The physical examination did not reveal any abnormalities. He had no liver, spleen, or lymph node enlargement. No rash on the skin or mucosa was noted.3/C4/C50), antistreptolysin O titer, antimitochondrial andibodies, anti-smooth muscle antibodies, anti nutrophilic cytoplasmic antibodies, anti-b2-glycoprotein-I antibody, and anticardiolipin antibodies all being normal. Cryoglobulins were also negative, as was the direct Coombs test. The chest X-ray was normal, as was the heart ultrasound. A PPD (purified protein derivative) skin test was performed and found negative.His complete blood count showed anemia, leucopenia and thrombocytopenia. He also had increased serum markers of inflammation, a marked hypergammaglobulinemia and a slightly affected renal and liver function along with a normal urinalysis Table He underwent an extensive imaging evaluation with a chest, abdominal and pelvic CT scan and a brain MRI, as well as a percutaneous liver biopsy that revealed a mild steatohepatitis.Finally, he had a bone marrow aspiration and biopsy that confirmed the diagnosis of myelodysplastic syndrome with 8% blasts   , liver  and renal function (Creatinine 1.6 mg/dL), and an abnormal urine sediment . A renal biopsy was scheduled in order to evaluate his nephritic syndrome, but was cancelled due to severe thrombocytopenia with positive antiplatelet antibodies.During his hospitalization he presented fever, a vasculitic rash at his neck, torso and arms (histopathologic examination showed a leukocytoclastic vasculitis), along with deterioration of his anemia (Ht = 26%), thrombocytopenia  resulting in rapid improvement of his clinical findings and biochemical profile. Methotrexate 10 mg/week PO was added to the regimen after 2 unsuccessful attempts of steroid tapering, while, due to persistent and severe pancytopenia, he was treated with a course 8 monthly cycles of 5-azacytidine in a dose of 75 mg/mFifteen months after initial diagnosis, bone marrow karyotypic and molecular testing  revealed no abnormalities. About 15 months after the first positive PCR for HTLV-1, and 11 months after initiation of azacytidine, a second test was carried out and was found negative.One month later, the patient underwent allogeneic peripheral blood stem cell transplantation from a fully matched unrelated donor with a once daily intravenous busulfan and fludarabine myeloablative conditioning regimen. A bone marrow aspirate on day +30 demonstrated trilineage engraftment and complete chimera (100% donor). On day +58 the patient developed grade II graft versus host disease (GVHD) of the skin that responded promptly to treatment with steroids. Currently, 8 months after transplantation, the patient remains in complete remission.The association of myelodysplasia with autoimmunity is well established for almost 20 years and has been confirmed by clinical and laboratory data ,6.There are several reports in the literature correlating various autoimmune manifestations such as leukocytoclastic vasculitis , relapsiMDSs have been linked to several types of glomerular diseases, such as glomerulosclerosis, membranous nephropathy, IgA nephropathy, and ANCA associated nephropathy -25. UnfoBoth the innate and adoptive immune system have been implicated in the autoimmune manifestations of patients with MDS in several studies evaluating T cell activation, function, and kinetics, as well as associations with the HLA system ,27.A large number of reports favor the use of immunoregulatory treatments  such as In our case, myelodysplastic syndrome and autoimmune phenomena were also accompanied by a positive PCR for HTLV-1. HTLV-1 has been implicated in the pathogenesis of ATLL and tropical spastic paraparesis. Although the virus is oncogenic, it neither induces oncogenesis by encoding oncogenes, nor does it integrate into the host genome to disrupt host gene expression. Rather, viral gene products interact with host proteins (mainly transcription factors) altering their function.It is estimated that 10-20 million people are infected by the virus worldwide. In endemic areas  seroprevalence ranges from 3% to 30%. In non-endemic areas, such as Greece, seroprevalence is less than 1%. Seropositivity among blood donors in Greece was reported to be 0.009% in a large multicenter study [Transmission of the virus is mainly achieved through breast feeding, sexual intercourse, inappropriate sterilization procedures and blood transfusion or injection drug use. Our patient claimed not to be an injection drug user, and had never been transfused in the past. His wife tested negative for HTLV-1.In the literature serologic positivity for the virus has been detected in patients with myelodysplastic syndromes in both endemic  and non Most studies about the prevalence of HTLV-1 in hematologic diseases come from Japan where the infection is endemic. Several case reports suggest the possible association between immune thrombocytopenia (ITP) and HTLV-1 infection . RecentlAssociation of the virus with rheumatologic manifestations is not rare and includes uveitis, chronic inflammatory arthropathy, Sjögren's syndrome, lymphocytic alveolitis, polymyositis, and fibromyalgia, but all these reports come from regions endemic for the HTLV-1 infection -40. MoreTreatment of the diseases caused by HTLV-1 is problematic. There have been only limited studies of specific antiretroviral therapy using nucleoside analogue reverse transcriptase inhibitors for HTLV-1 infection -45.Azacytidine and its deoxy derivative, decitabine (5-aza-2'deoxycytidine), are FDA-approved agents for the treatment of low and high risk patients with MDS. Azacytidine is incorporated into DNA and RNA by methyltransferases and acts as a false substrate and a potent inhibitor of methyltransferases. This results in reduction of DNA methylation (hypomethylating or demethylating agent) affecting the binding of cell regulation proteins to the DNA/RNA substrate ,47. DNA It has been shown that 5-azacytidine inhibits human immunodeficiency virus type 1 (HIV-1) replication . This anThe antiretroviral activities of hypomethylating factors, such as decitabine and 5-azacytidin are the subject of several current studies. One of them suggests a more specific role of 5-azacytidine in the treatment of ATLL, as it shows a growth inhibition of leukemic cells, offering a potential new therapeutic approach to improve the poor outcomes associated with ATLL .Although in our case, HTLV-1 negativity may have resulted from other unidentified factors, such as immune restoration following treatment, our case may be another example of drug repositioning, were azacitidine administered to treat MDS, had also an effect on HTLV-1 replication, resulting into a negative PCR for the virus several months following treatment.This is a unique case of a patient with RAEB-1, accompanied by severe autoimmune manifestations, that was infected with HTLV-1 in a region non-endemic for the virus. The patient achieved a complete remission of his hematologic disease and autoimmune manifestations after treatment with 5-azacytidine, while several months later he tested negative for HTLV-1 by nested PCR, a fact that can be linked to the, already identified by other studies, antiretroviral action of the drug.Written informed consent was obtained from the patient for publication of this Case Report.The authors declare that they have no competing interests.PTD, Acquisition analysis and interpretation of data, drafting of the manuscript and revising the manuscript. MM, Acquisition and analysis of data. OB, Revising the manuscript. EB, Acquisition and analysis of data. GT, Acquisition and analysis of data. JM, Revision of the manuscript. GV, Revision of the manuscript. NAV, Final revision and approval of the manuscript.


SATB2 causes severe defects to the palate and jaw. The expression and sequence of SATB2 is highly conserved across vertebrate species, including zebrafish. We sought to understand the regulation of satb2 using the zebrafish model system. Due to the normal expression domains of satb2, we analyzed satb2 expression in mutants with disrupted Hh signaling or defective ventral patterning. While satb2 expression appears independent of Edn1 signaling, appropriate expression requires Shha, Smo, Smad5 and Hand2 function. Transplantation experiments show that neural crest cells receive both Bmp and Hh signaling to induce satb2 expression. Dorsomorphin- and cyclopamine-mediated inhibition of Bmp and Hh signaling, respectively, suggests that proper satb2 expression requires a relatively earlier Bmp signal and a later Hh signal. We propose that Bmp signaling establishes competence for the neural crest to respond to Hh signaling, thus inducing satb2 expression.In human, mutation of the transcription factor  Cranial neural crest cells are a highly specialized vertebrate-specific cell type which give rise to diverse structures including most of the bone and cartilage of the head, face and palate. In all vertebrates, this pluripotent cell population migrates to the periphery in three streams and condenses within transient structures known as pharyngeal arches Edn1 show reduced expression of genes known to be essential for mandibular development such as Hand2Pharyngeal arches consist of a mesodermal core surrounded by neural crest cell-derived mesenchyme that is encapsulated by epithelia (the ectoderm and the endoderm). Reciprocal interactions between these different cell types are critical to establish the signaling networks required to generate the multitude of craniofacial elements of the appropriate size and shape SHH or its downstream effectors result in a variety of severe developmental disorders; those affecting the head and face include holoprosencephaly, cyclopia and hypotelorism . Neural crest cells must receive Hh signaling for the appropriate expression of patterning genes in the developing craniofacial skeleton Shh is a member of the secreted family of Hedgehog (Hh) proteins and all hedgehog signaling is transduced through Smoothened (Smo). Shh is crucial for regulating patterning and outgrowth of the face and palate across vertebrate species Considering the numerous transcription factors and signaling molecules involved in regulating craniofacial development it is not surprising that craniofacial defects are common among congenital birth defects. The most common craniofacial defects are cleft lip and/or palate and mandibular dysmorphogenesis satb2 is a target of Bmp and Hh signaling. Using genetic mosaics, we find that neural crest cells must receive both signals for proper satb2 expression. Using pharmacologic inhibition of each pathway, we find that Bmp signaling is required temporally before Hh signaling. Collectively, we provide some of the first evidence regarding the regulation of satb2.Here, we use zebrafish to gain insight into how Satb2 integrates into the signaling cascades involved in craniofacial development. We show that Danio rerio) embryos were raised at 28.5°C and staged as described previously in situ hybridization analysis were incubated in embryo media containing PTU to prevent pigmentation. Cyclopamine experiments y1tg line, designated fli1:EGFP for clarity mw40tg(BRE-AAVmlp:dmKO2) line, designated BRE:mKO2BRE:mKO2 expression at the time embryos were fixed for in situ hybridization. The exact same settings were used for the collection of all BRE:mKO2 images. t4shhab577smotf216bedn1s6hand2ta56sox32edn1 and hand2 mutant embryos were kind gifts from The University of Oregon aquarium. Heterozygous carriers of the smoothened null allele, smo, and sox32 allele fli1:EGFP background and are referred to as -smo and −sox32 in the text for clarity. The b1100smad5smad5. This mutation is predicted to generate a Proline to Serine missense mutation in the DNA binding MH1 domain of Smad5. PCR with primers jaa1 (5′ aagggcctcccacacgtcatct 3′) and yj32 (5′ ctggactttcaactcgtttgtagtgccatg 3′), followed by digestion with NcoI was used for genotyping. The mutant allele is 160 bp and the wt allele is 129 bp.The study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee at the University of Texas at Austin (AUP-2012-00053). Zebrafish  Untreated 30 hpf BRE:mKO2 embryos have rhobust transgene expression in the ventral pharyngeal arches (arrows). (B) Nearly all expression, except for some in the dorsal retina is lost following dorsomorphin treatment from 20–30 hpf. (C) At 36 hpf the expression of the BRE:mKO2 transgene closely resembles that observed at 30 hpf. (D) Embryos treated with dorsomorphin from 20–30 hpf and then washed out of the drug show a partial recovery of BRE:mKO2 expression in the ventral pharyngeal arches (arrows).(TIF)Click here for additional data file.


Approximately 10 to 15% of couples are impacted by infertility. Recently, the pivotal role that lifestyle factors play in the development of infertility has generated a considerable amount of interest. Lifestyle factors are the modifiable habits and ways of life that can greatly influence overall health and well-being, including fertility. Many lifestyle factors such as the age at which to start a family, nutrition, weight, exercise, psychological stress, environmental and occupational exposures, and others can have substantial effects on fertility; lifestyle factors such as cigarette smoking, illicit drug use, and alcohol and caffeine consumption can negatively influence fertility while others such as preventative care may be beneficial. The present literature review encompasses multiple lifestyle factors and places infertility in context for the couple by focusing on both males and females; it aims to identify the roles that lifestyle factors play in determining reproductive status. The growing interest and amount of research in this field have made it evident that lifestyle factors have a significant impact on fertility. It has been estimated that 7.4% of women and their husbands in the United States are infertile  and thatThe goal of this review is to demonstrate the potential effects of multiple lifestyles on reproductive health for both men and women. The review focuses primarily on modifiable lifestyles including the age when starting a family, nutrition, weight management, exercise, psychological stress, cigarette smoking, recreational drugs use, medications, alcohol use, caffeine consumption, environmental and occupation exposure, preventative care, clothing choices, hot water, and lubricants. While many aspects of life are not modifiable, lifestyles may be changed.The age of a man or woman is a factor among others that can affect fertility. Due to pursuit of education and other factors, many couples are choosing to delay child-bearing. Fertility peaks and then decreases over time in both men and women, thus the reproductive timeline may be one aspect to consider when determining the ideal time to start a family. As men age, testosterone levels begin to decrease and hypogonadism results. However, if testosterone is used to treat hypogonadism, it can suppress spermatogenesis . Semen pThe reproductive timeline for women is complex. A woman is born with all the oocytes she will ever have, and only 400–500 are actually ovulated . As the Eating a healthy and varied diet may be a key part of maintaining good overall health. However, there are certain vitamins and food groups that could have a greater impact on reproductive health than others.2• -), hydrogen peroxide (H2O2), hydroxyl radical (OH•). This category also includes free radicals derived from nitrogen called reactive nitrogen species such as: nitric oxide (NO•), nitric dioxide (NO2•), peroxynitrite (ONOO-). Collectively they are termed as reactive oxygen species. These are by-products of cellular respiration that are necessary for certain cellular activity, including sperm capacitation; however, an overabundance of ROS may compromise sperm function, including sperm motility, altering DNA and decreasing membrane integrity [Aspects of a male’s diet may have an impact on his fertility. Consuming a diet rich in carbohydrates, fiber, folate, and lycopene  as well ntegrity . Antioxintegrity . Howeverntegrity . A high ntegrity . Anotherntegrity . Suleimentegrity . An artintegrity . A Cochrntegrity . Antioxintegrity .A woman’s diet may ultimately affect her fertility, particularly ovulation. Overall, replacing carbohydrates with animal protein was demonstrated to be detrimental to ovulatory fertility (OR 1.18) . Adding An individual’s weight is often associated with his or her eating habits and amount of activity. Body mass index (BMI) is reported as a number. If it is below 18.5 it is considered underweight, between 18.5 and 24.9 is normal, above 25 is overweight, and over 30 is considered obese . Body weThe obesity epidemic has recently become is a serious issue, particularly in industrialized nations. The goal set by Healthy People 2010 of reducing obesity in the United States to 15% was not met . In factThe proportion of men over 20 years of age in the U.S. that are obese has risen to 35.5% . BMI mayA relationship also exists between obesity and erectile dysfunction (ED). Corona et al. reported that 96.5% of men with metabolic syndrome presented with ED n = 236) . ED may  . ED may2 = .03, P = .008) [In 2010, 35.8% of women in the U.S. over the age of 20 were considered obese . Women w = .008) . A decre = .008) . There i = .008) . The neg = .008) .Obesity is not the only way in which weight can impact fertility. Men who are underweight are also at risk of infertility. Men who are underweight tend to have lower sperm concentrations than those who are at a normal BMI . As the For women, being underweight and having extremely low amounts of body fat are associated with ovarian dysfunction and infertility . AdditioA healthy amount of exercise in men can be beneficial. Physically active men who exercised at least three times a week for one hour typically scored higher in almost all sperm parameters in comparison to men who participated in more frequent and rigorous exercise (n = 45) . ModeratPhysical activity has been shown to confer a protective effect on fertility when coupled with weight loss in obese women . HoweverStress is a prominent part of any society, whether it is physical, social, or psychological. Infertility itself is stressful, due to the societal pressures, testing, diagnosis, treatments, failures, unfulfilled desires, and even fiscal costs with which it is associated .Males who experienced more than two stressful life events before undergoing infertility treatment were more likely to be classified below WHO standards for sperm concentration, motility, and morphology . In a stStress can increase after diagnosis of infertility, follow-up appointments, and failed IVF treatments . When mePhysical stress has been implicated in influencing female fertility. Women who had a job and worked more than 32 hours a week experienced a longer time to conception compared to women who worked 16 to 32 hours a week . PsycholReceiving instruction on how to deal effectively or merely receiving support made a significant difference for women undergoing fertility treatment. There was a higher conception rate for women who were part of a cognitive behavioral intervention group (55%) or a support group (54%) than for those women who were not receiving any intervention (20%) . Women wWhile it is well documented that cigarette smoke contains over 4,000 chemicals  and is aAmong women who are of reproductive age, 30% are smokers . Augood Studies of the effects of illegal drugs on human fertility have been scarce due to ethical considerations, as well as subject to under-reporting and bias due to the characteristics of the population being studied, such as low socioeconomic status or improper prenatal care . Use of Marijuana is one of the most commonly used drugs around the world , and it Another commonly used recreational drug is cocaine, a stimulant for both peripheral and central nervous systems which causes vasoconstriction and anesthetic effects. It is thought to prevent the reuptake of neurotransmitters , possiblOpiates comprise another large group of illicit drugs. Opiates, such as methadone and heroin, are depressants that cause both sedation and decreased pain perception by influencing neurotransmitters . In men In general, there are more studies reviewing the effects of medication on male than female fertility. It is necessary to first determine which medications cause fertility issues, and to then determine if these effects are permanent. A study headed by Hayashi, Miyata, and Yamada investigated the effects of antibiotics, antidepressants, antiepileptics, β stimulators, H1 and H2 receptor antagonists, mast cell blockers, and sulfonylurea compounds (n = 201) . Male paMany studies have been conducted on the effects of alcohol and aspects of health, including fertility. While there are studies that demonstrate the link between alcohol and infertility, it is not entirely clear what amount relates to an increased risk.2) n = 35) [In men, alcohol consumption has been linked with many negative side effects such as testicular atrophy, decreased libido, and decreased sperm count -113. One n = 35) . A link  n = 35) . In addi n = 35) . While a n = 35) ,115, but n = 35) .Women who drink large amounts of alcohol have a higher chance of experiencing an infertility examination than moderate drinkers  in comparison to those who consumed low amounts, who had a decreased chance of experiencing an infertility examination   . A commoCaffeine has become an integral part of society with consumption varying from 50 mg in a 16 oz. bottle of Pepsi to 330 mg in a 16 oz. cup of Pikes Place Roast from Starbucks ,123. HowMany potential threats to reproductive health are encountered in every-day life through biological (viruses), physical (radiation), and toxic  sources . While tAir pollution is the release of pollutants such as sulfur dioxides, carbon monoxide, nitrogen dioxide, particulate matter, and ozone into the atmosphere from motor vehicle exhaust, industrial emissions, the burning of coal and wood, and other sources ,133. WhiThere have been reports of air pollution and its impacts on male fertility. Several studies have been conducted in the Czech Republic regarding men living in two different locations, one more polluted than the other ,135. MenNegative reproductive side effects of air pollution on women can include preterm delivery ,137, misHeavy metals include metals such as lead, mercury, boron, aluminum, cadmium, arsenic, antimony, cobalt, and lithium. Only a few such heavy metals have been researched in connection to reproductive function. Lead, which is commonly found in batteries, metal products, paints, ceramics, and pipes, is one of the most prominent heavy metals. Lead interrupts the hypothalamic-pituitary axis and has been reported to alter hormone levels ,138, altMany of the chemicals used world-wide in today’s society, including pesticides and endocrine disruptors, among others, may have various damaging effects on the reproductive health of both men and women. Mimicking natural hormones, impeding normal hormone activity, and varying regulation and function of the endocrine system are a few of the many ways that endocrine disruptors influence one’s body . NumerouBoth men and women can be exposed to chemicals and other materials that may be detrimental to their reproductive health while on the job. Heavy metals and pesticides, as outlined in Table Exposure to various kinds and amounts of radiation can have lasting effects in humans. Radiation that is in the form of x-rays and gamma rays can be devastating to the sensitive cells of the human body, including germ and Leydig cells. The damage done depends on the age of the patient and dose, and ultimately can result in permanent sterility ,152.2 to 9.2 ± .7 μm2 and acrosomal area significantly decreases from 21.5 ± 4% to 35.5 ± 11.4% (P < .05) [The incredible convenience of the cell phone has dramatically increased its usage in the last decade. However, it does not come without negative effects. There have been an increasing number of studies demonstrating negative effects of the radiofrequency electromagnetic waves (RFEMW) utilized by cell phones on fertility. Cell phone usage has been linked with decreases in progressive motility of sperm , decreasP < .05) . In addiP < .05) . While aP < .05) ,158. AnoP < .05) , providiWhile contraceptives are often associated with preventing pregnancy, several studies have demonstrated that both condom usage and oral contraceptives can preserve fertility in women ,160. In Scheduling regular doctor appointments may be beneficial for fertility. Males tend to not seek medical treatment for sexual dysfunctions or infections. It was reported that when men experience sexual problems, only 10.5% seek help n = 11,161) . When th . When tFor women, visiting the gynecologist to receive an annual pap smear has been associated with being protective of fertility  . Kelly-WThe type of clothing a man chooses to wear, may have effects on reproductive health. Many studies have been conducted hoping to find an answer to the question of what type of clothing is best for fertility. The view that elevation of scrotal temperature negatively impacts spermatogenesis and sperm parameters is universally acknowledged . But theOne study followed 20 participants who wore tight-fitting underwear for 6 months then switched to loose-fitting underwear for 6 months . Semen sLiterature providing evidence that wet heat is tied to infertility is scarce. Many fertility authorities rely on the data provided from research of the effects of temperature on sperm function and then apply the idea to hot baths, jacuzzis, or saunas. One study conducted by Shefi et al. actually studied the effects of wet heat on 11 male subjects who were exposed to wet heat for greater than 30 minutes every week for at least 3 months prior to any experimentation . These 1Many sexually active couples choose to utilize vaginal lubricants to treat vaginal dryness and pain during intercourse . While aLifestyle factors, including age when starting a family, nutrition, weight management, exercise, psychological stress, cigarette smoking, recreational and prescription drugs use, alcohol and caffeine consumption, environmental and occupational exposures, preventative care, and other behaviors are modifiable and may impact fertility.The evidence suggests that age may play a large role in determining fertility. Attempting pregnancy before the age of 30 for women and before 35 for men may provide the highest chances of success. While it is important for one partner to consider their age, it is when both partners consider their ages together that they may be able to thoroughly increase their odds of having a successful pregnancy.Proper nutrition, weight, and exercise may impact fertility. Though no definitive link has been drawn, choosing proper nutrition, whether it be choosing supplements or food groups, before and during attempts to conceive may be vital for improving fertility for both men and women. Men and women who are underweight or overweight are also at risk for negative side effects, including changes in hormone levels that heavily influence their fertility. Recent research suggests that weight plays an important role in fertility, and controlling and maintaining an ideal weight may provide a way for couples to increase their fertility. Exercise is suggested to be beneficial, though too much may be detrimental. Lean and underweight men or women who exercise vigorously may put themselves at risk for a decrease in fertility, thus finding a balance may provide the best chances of achieving a pregnancy.While there are associations between psychological effects and infertility, it is hard to establish a cause-effect relationship. Tests are subjective, and there is no general consensus on how to measure psychological stress . It is aRecreational and prescription substances also appear to have significant impact on fertility. Though clear links are yet to be determined, there are negative trends, including decreased semen parameters or decreased ovarian reserve, associated with smoking and fertility. If couples are attempting to achieve a pregnancy, limiting or smoking cessation may provide more positive outcomes. It is difficult to establish a link between illegal drug use and infertility as there are many ethical issues that prevent researchers from discovering a definitive relationship. Researchers have also had issues with drawing a definitive link between infertility and prescription medication, which often have known side effects, though fertility side effects are not often a concern when prescribing. Further research on many medications and drugs is needed in order to make any recommendations. While there is evidence to support that alcohol does have an impact on fertility, it is also difficult to establish a definitive link as there is no standard “drink” or comparative way to measure alcohol consumption. Despite these drawbacks to recent research, decreasing or ceasing alcohol consumption may provide a better chance of achieving a viable pregnancy for the couple. Though caffeine appears to have a negative effect on fertility, additional research is needed to elucidate if there is a definitive relationship.Concerning environmental exposures, assessing the exposures of each individual may be crucial to reproductive health of the couple. Eliminating every exposure is unrealistic; however, identifying, eliminating, or minimizing even one factor may have significant positive effects on fertility for both men and women.Taking care of a current fertility problem may provide better fertility in the future. Taking preventative steps such as visiting your doctor and using contraception may help fertility. Making appointments with a doctor for both preventative measures and when problems arise may assist in increasing fertility for both men and women. In addition, using appropriate contraception may have a positive impact on a couple’s fertility. Other factors such as clothing choice, wet heat, and lubricants may also impact a couple’s fertility. Overall, while there is suggestive data, a clear negative influence of the type of underwear or clothing on semen quality has not been proven, and the overall effects on a couple’s fertility are still unknown. While there is suggestive data, there is very little actual evidence linking wet heat and fertility to suggest cessation of the use of wet heat. Research suggests that some lubricants can be beneficial for couples trying to conceive, and some lubricants may be detrimental to fertilization. Choosing an appropriate lubricant may provide the couple with improved chances of achieving a pregnancy.The lifestyle factors discussed in the present review have the potential to impact fertility. It is important to understand the ways in which lifestyle behaviors may benefit or harm fertility in order to minimize complications and to maximize fertility outcomes. By understanding the impact of lifestyle on reproductive health, and by actively modifying lifestyle behaviors, men and women are capable of controlling their own fertility potential.OR: Odds ratio; ROS: Reactive oxygen species; IUI: Intra uterine insemination; MDA: Malondialdehyde; RR: Relative risk; BMI: Body mass index; ED: Erectile dysfunction; SHBG: Sex Hormone-Binding Globulin; GnRH: Gonadotropin-Releasing Hormone; LH: Luteinizing Hormone; HA: Hyaluronan; TAC: Total antioxidant capacity; FSH: Follicle stimulating hormone; RFEMW: RadioFrequency ElectroMagnetic Waves; BPA: Bisphenol A; DDT: DichloroDiphenylTrichloroethane; DDE: DichloroDiphenyldichloroEthylene; 5-ASA: 5-Aminosalicylic acid.The authors declare that they have no competing interests.RKS conceived the study, participated in the study design compilation of the contents and critical review of the paper. KRB and JMF were responsible for literature (Medline) search, compilation of the information, drafting and finalizing the paper. AA provided substantial contribution ranging from study idea, design, and critical review of the final paper. All authors read and approved the final manuscript.KRB and JMF were summer interns at the Center for Reproductive Medicine, Glickman Urological & Kidney Institute, Cleveland Clinic, Cleveland, Ohio. RS is the Coordinator of the Center for Reproductive Medicine, Cleveland Clinic, Cleveland Ohio. AA is the Director of the Center for Reproductive Medicine, Cleveland Clinic, Cleveland Ohio.


HTLV-1 molecular epidemiological studies demonstrated its great genetic stability, with different subtypes/subgroups, linked to the geographical origin. These peculiar features can be used as molecular means to study migrations of HTLV-1 endemic populations. Although the Caribbean archipelago is a high HTLV-1 endemic area, little is known concerning the HTLV-1 genetic diversity in this region.HTLV-1 infected persons, either patients or HTLV-1 asymptomatic carriers, mostly living in Paris area, were included. Their geographical origin was determined through interviews. DNAs, extracted mostly from PBMCs were subjected to PCRs amplifying two LTR fragments. PCR products were sequenced and phylogenetically analyzed.112 persons were included with 55 from West-Indies, 26 from French Guyana, 23 from West Africa and 8 from Central Africa. Comparison of the complete LTR indicated that most of the West African strains were of the Senegal subgroup or West African one (AWA). In contrast and surprisingly, all West Indian strains, except one, belong to the Transcontinental (TC) subgroup of the Cosmopolitan subtype. Interestingly, half of the French Guyanese were infected by a TC strain while the others  were infected by an AWA strain.Further studies combining a larger number of samples from West Indian and West Africa, together with precise historical data are ongoing to gain better insights into the origin and modes of dissemination of HTLV-1 in the Caribbean and surrounding areas, as well as the origin of these populations in Africa.


Although there are significant numbers of people displaced by war in Africa, very little is known about long-term changes in the fertility of refugees. Refugees of the Mozambican civil war (1977–1992) settled in many neighbouring countries, including South Africa. A large number of Mozambican refugees settled within the Agincourt sub-district, underpinned by a Health and Socio-demographic Surveillance Site (AHDSS), established in 1992, and have remained there. The AHDSS data provide a unique opportunity to study changes in fertility over time and the role that the fertility of self-settled refugee populations plays in the overall fertility level of the host community, a highly relevant factor in many areas of sub-Saharan Africa.To examine the change in fertility of former Mozambican self-settled refugees over a period of 16 years and to compare the overall fertility and fertility patterns of Mozambicans to host South Africans.Prospective data from the AHDSS on births from 1993 to 2009 were used to compare fertility trends and patterns and to examine socio-economic factors that may be associated with fertility change.There has been a sharp decline in fertility in the Mozambican population and convergence in fertility patterns of Mozambican and local South African women. The convergence of fertility patterns coincides with a convergence in other socio-economic factors.The fertility of Mozambicans has decreased significantly and Mozambicans are adopting the childbearing patterns of South African women. The decline in Mozambican fertility has occurred alongside socio-economic gains. There remains, however, high unemployment and endemic poverty in the area and fertility is not likely to decrease further without increased delivery of family planning to adolescents and increased education and job opportunities for women. Africa is home to about a fifth of the world's refugees, most of whom have been victims of forced migration .‡ HoweveWar and resettlement can place both upward and downward pressure on fertility in the short term. Upward pressure may come from the desire to replace those lost in war, while downward pressure on fertility may come from the disruption of life and relationships caused by war . StudiesHowever, most studies on refugee fertility are conducted in refugee camps and the situation may differ for refugees not living in camps. Populations that settle in host countries without residing in camps are likely to be different from those in refugee camps since they are not served directly by aid programs. Many studies of the fertility of self-settled refugees exist in developed countries with vital registration systems. However, studies of self-settled refugee populations in Africa where vital registration systems are lacking are rare. Prospective data from the Agincourt sub-district in Mpumalanga Province in rural northeast South Africa provide an opportunity to examine the change in fertility of self-settled Mozambican refugees over a period of 16 years (1993–2009) and to examine their impact on overall fertility levels in the area. Earlier research using data from the Agincourt health and socio-demographic surveillance site (AHDSS) found that Mozambican refugees in Agincourt contributed to a noticeable increase in the average number of children borne  by women in the 1980s measured retrospectively through birth histories . Subsequapartheid regime to the spectre of rapid population growth among the African population. The programme provided free modern contraceptives in public health clinics, including oral and injectable contraceptives , Gazankulu, where African South Africans were resettled as part of the apartheid regimes strategy of ‘separate development’  and the level of fertility measured by the TFR are used to examine fertility trends. The latter is defined as the average number of children that a woman would have by the end of her reproductive life if the current age pattern of fertility were to remain unchanged. Descriptive statistics are used to describe changes in the age pattern of fertility over time. A discrete time event framework is used to evaluate women's progression from a first to a second birth within five years and smoothed survival curves are presented. Other socio-economic trends are examined by estimating levels of employment, household wealth, and formal education.Fertility levels were quite different in the two populations during the 1990s, with Mozambican women maintaining higher fertility than South Africans. Thereafter, the two populations increasingly exhibit similar fertility levels, converging from 2000 when the confidence intervals around the fertility estimates for the two groups started overlapping. The convergence of total fertility of the two population groups is driven primarily by the decline in fertility among Mozambican women to the levels of South African women. This suggests that Mozambican women were adopting fertility behaviours similar to those of the host population. To test this hypothesis we compared age-specific fertility rates and the timing of first and second births between the two populations at the beginning and end of the observation period.However, by 2009, Panel B of The age-specific fertility rates suggest similarly high levels of adolescent fertility for Mozambican and South African women. Further analysis of the age distribution of first births for Mozambican women  shows inFurther analysis also suggests lower contraceptive use by Mozambican women prior to their first birth. At the time of their first birth, Mozambican women consistently reported lower contraceptive use prior to conceiving than South African women. Five per cent of Mozambican women compared to 9.5% of South African women with first births from 1995 to 1999 reported using contraception at some time before their first birth. These figures were 23% and 28%, respectively, for first births occurring from 2003 to 2005. High adolescent fertility has been a source of concern in South Africa and so it is important to recognise the lower use of contraception before a first birth as well as the increase in the percentage of first births to adolescents for Mozambican women .Previous research on the fertility of host South Africans has shown that fertility decline for African South Africans has been driven by significant widening of birth intervals explained primarily by increases in the use of modern contraception . Wide biThe changes in age-specific fertility rates, timing of first births and extended first birth intervals indicate that Mozambican women are achieving lower fertility by adopting patterns of childbearing typical for South African women in Agincourt.To further explore the fertility decline and the convergence of fertility in the two populations, we examine select socio-economic factors that may be ‘underlying’ drivers of the decline in the TFR among the Mozambicans. Increases in education, labour force participation and income have been found to reduce fertility –22. HistPanel B of An analysis of the labour force participation of women of reproductive ages shows that formal employment increased slightly during the past decade for South Africans (from about 28% in 2000 to 30% in 2008) but decreased for Mozambicans (from about 27% to 23% over the same period). The very high unemployment of both groups suggests limited formal economic opportunities for women, which might have contributed to the recently observed stall in fertility decline.Education and wealth indicators suggest that over the period of study Mozambican women's status improved and converged with that of South African women. However, these gains are relatively modest and Mozambican women remain disadvantaged, particularly in relation to formal employment, within the relatively poor population of the rural setting.Approximately 20 years after the civil war in Mozambique, demographic characteristics of self-settled refugees of Mozambican origin in Agincourt are converging with those of their South African hosts. While the TFR in Mozambique itself has remained near 5 , the MozThe findings of this study suggest adaptation of the Mozambican refugees in the AHDSS to the fertility patterns of their host community. Adaptation theory states that exposure to cultural norms and local costs of childbearing will lead migrants to change their fertility behaviour to converge with that of natives in the destination . This apThe adaptation of Mozambican refugees to the lower fertility regime in South Africa has important implications for many areas of sub-Saharan Africa hosting refugee populations. The adaptation of Mozambicans in South Africa is likely facilitated by a shared language and culture. Self-settled refugees are also probably more likely to be exposed to and adjust to the local norms of childbearing compared to refugees living in camps.Access to contraception through the South African health system is a key component of the decrease in fertility of Mozambicans. Another important component is the improvement in socio-economic status partly attributable to access to education and host government social grants. Reducing the economic disadvantage of refugees and integrating refugees into local programmes and services encourages adaptation and can compensate for other factors that may otherwise increase the fertility of refugees such as poverty, lack of education, and lack of reproductive health services. Integration encourages adaptation and will likely benefit host communities by lowering the fertility of refugees.Overall fertility decline in Agincourt over the past few decades has been driven primarily by the decline in fertility of Mozambican women. South African women's total fertility declined primarily in the early 1990s and has been wavering around 2.5 since 1995. Fertility decline has also been minimal for Mozambican women since 2002. With fertility decline stalling in both groups it remains to be seen if fertility will go below replacement level (2.5 in South Africa) as predicted by earlier research . FurtherFindings presented here suggest a few areas of future intervention that would be helpful in settings such as Agincourt. The pattern of childbearing in Agincourt shows that delaying first births could reduce overall fertility rates. Others have argued that family planning programmes in South Africa need to be reoriented to address the contraceptive needs of adolescents before first births . Since cIn other settings, increasing access to family planning and reproductive health programmes for all women has been shown to improve women's economic and health outcomes and to enhance economic growth . HoweverThe primary limitations of our study are data driven. We do not have information on important variables such as prospective data on marriage, fertility desires, or detailed information on contraceptive use, to run models examining the proximate determinants of fertility.


HTLV-1 is a retrovirus that causes lymphoproliferative disorders and inflammatory and degenerative diseases of the central nervous system in humans. The prevalence of this infection is high in parts of Brazil and there is a general lack of public health care programs. As a consequence, official data on the transmission routes of this virus are scarce.To demonstrate familial aggregation of HTLV infections in the metropolitan region of Belém, Pará, Brazil.A cross-sectional study involving 85 HTLV carriers treated at an outpatient clinic and other family members. The subjects were tested by ELISA and molecular methods between February 2007 and December 2010.P = 0.0033). Sexual and vertical transmission was likely in 38.3% (23/60) and 20.4% (29/142) of pairs, respectively . Positivity was 51.3% (20/39) and 14.3% (3/21) in wives and husbands, respectively . By age group, seropositivity was 8.0% (7/88) in subjects <30 years of age and 36.7% (51/139) in those of over 30 years . Positivity was 24.1% (7/29) in the children of patients infected with HTLV-2, as against only 5.8% (4/69) of those infected with HTLV-1 .The prevalence of HTLV was 43.5% (37/85) for families and 25.6% (58/227) for the family members tested  has a slow replication rate and infection is characterized by low morbidity and mortality, as well as silent transmission within the population. While rare, HTLV-associated diseases are usually debilitating and life-threatening. The virus is endemic in the state of Pará (Brazil), although there have been no studies of the distribution of the virus within the local population. The results of the present study confirm the existence of familial aggregations of HTLV infection in the metropolitan region of the state capital, Belém. Considerably higher rates of sexual transmission of HTLV from men to women were also demonstrated. Rates of infection were similar for the two virus types, although HTLV-2 appears to be increasing in the population. The frequency of positivity among family members increased in direct proportion to age and was associated with a relatively large proportion of asymptomatic carriers. In addition, widespread ignorance of the virus increases the risk of transmission. The available evidence indicates that significant human suffering is caused by this virus in patients suffering complications, and this is little justification for the lack of intervention on the part of public health authorities, which might impede the ongoing proliferation of this infection in the population. The acronym HTLV is the internationally accepted abbreviation for human T-cell lymphotropic virus, which belongs to the family Retroviridae. This virus has been implicated as the etiological agent of lymphoproliferative disorders, inflammatory and degenerative diseases of the central nervous system, and some immune system disorders About 20 million people are estimated to be infected with HTLV worldwide Studies of HTLV transmission in endemic regions worldwide have indicated that the virus spreads silently, primarily from mother to child and through sexual contact Between June, 2007, and December, 2010, blood samples were collected from relatives of the 85 HTLV outpatients being monitored at the clinic of the Infectious Diseases Service in Belém, Pará, Brazil, who agreed to participate in the study. All 85 of these cases had been diagnosed by detection of proviral HTLV-1 or HTLV-2 DNA and lived in the metropolitan region of Belém.The inclusion of contact subjects (N = 227) was based on the criterion of family relationships, given known HTLV transmission routes. Given, samples were solicited from the spouse and mother of male subjects, and the mother, spouse and children of female subjects. Other family members were recruited based on the identification of HTLV-positive cases among the contacts already investigated, using the same selection criteria.Each family member was tested for HTLV-1/HTLV-2 antibodies by ELISA . All seropositive relatives were tested for Polymerase Chain Reaction/Restriction Fragment Length Polymorphism (PCR/RFLP) for the confirmation of infection and the identification of the viral genotype For analysis, the cases were divided into groups according to virus type (HTLV-1 and HTLV-2), symptoms (symptomatic or asymptomatic), and transmission route . Cases with symptoms not related to diseases caused by HTLV and those with no or few symptoms were classified as asymptomatic. The symptomatic patients sought health care most frequently and most often presented symptoms consistent with injuries caused by HTLV, based on the criteria reported in the literature.Variation related to gender, degree of kinship, virus type, and symptoms were evaluated statistically using Chi-square or Fisher's exact test, where appropriate, and Mann-Whitney's U. A 5% significance level was considered for all analyses. All the information collected during the interviews was stored in a DBMS MS Access 2007 database for processing and analysis. Access to this database was restricted to the authors of this study. All statistical analyses were run in the BioEstat 5.0 program This study complied with the principles outlined by the Declaration of Helsinki and was approved by the Human Research Ethics Committee of the Tropical Medicine Center of the Federal University of Pará. An informed consent form was signed by all the participants or their parents/legal guardians prior to the collection of samples and the clinical examinations.Sixty-two (72.9%) of the 85 outpatients infected with HTLV at NMT were infected with HTLV-1 and 23 (27.1%) with HTLV-2. Forty-seven (55.3%) were female and 38 (44.7%) male. Mean age at the time of enrollment at the clinic was 43.1 years (SD ±12.9), ranging from 12 to 64 years. Most cases (76.5%) were aged between 30 and 60 years.Strongyloides stercoralis and some patients presented two or three symptoms.Overall, 37 (43.5%) of the HTLV outpatients were classified as symptomatic and 48 (56.5%) as asymptomatic. Symptoms were only seen in patients infected with HTLV-1 and included neurological signs in 21 subjects, cutaneous manifestations in 16, rheumatological symptoms in 14, and ocular manifestations in 11. Five patients were infected with A total of 227 relatives were screened for HTLV infection by serological examination, including 154 (67.8%) females and 73 (32.2%) males, with a mean age of 37.3 years (SD ±18.2), ranging from one to 83 years. Slightly more than half of these subjects  reported a family income equal to or less than the minimum wage, 41% (93/227) an income of two to five times the minimum wage, and the remaining 7.9% (18/227) an income of more than five times the minimum wage. Education levels were also distributed bimodally – while 48.9% (111/227) of the subjects were high school graduates, 30.4% (69/227) were illiterate. Of the remainder, 16.7% (38/227) had completed primary school and 4% (9/227) were college graduates.P = 0.0033).Familial transmission of HTLV was observed in 37 of the 85 families investigated, of which, 43.5% had at least two infected members. Individual transmission was observed in 58 (25.6%) of the 227 subjects studied .The ages of seropositive subjects ranged from 6 months to 90 years (mean ± SD = 45.4±17.8 years), whereas those of seronegative individuals were between 2 and 83 years, mean = 33.5±18.2 years . SeroposSeroprevalence of HTLV was 29.2% (45/154) in female relatives versus 17.8% (13/73) in male relatives . The anaP = 0.0130).Vertical and sexual transmission occurred in 24 (28.2%) and 23 (27.1%) of the 85 families, respectively. Vertical transmission apparently occurred in 29 (20.4%) of 142 mother-son/daughter pairs, while sexual transmission probably occurred in 23 (38.3%) of 60 couples  or mother to son, 21.1% or 11/52 . The meaP = 0.0143). The seroprevalence rates observed in mothers of index cases were 46.4% (13/28) for HTLV-1 and 22.2% (2/9) for HTLV-2 .The seroprevalence in children of patients infected with HTLV-2 and HTLV-1 was 24.1% (7/29) and 5.8% (4/69), respectively , given the reduced sample size in the latter . All the couples reported maintaining a stable relationship. The average length of these relationships was 10.4 years in the cases where the spouses were seronegative, and 18.2 years in those in which they were seropositive.The prevalence of HTLV in wives  was significantly higher than that recorded in the husbands  of infected patients . SimilarP = 0.0117). HTLV-1 infection was identified in five (55.56%) of nine members of a family in which there were three probable routes of vertical transmission and one probable route of sexual transmission, as well as two deaths by non-Hodgkin lymphoma  of the 163 subjects  attended the center. The sample of 85 confirmed cases analyzed in the present study does not necessarily represent the prevalence of infection in the region, but reflects the reality of infection rates in the metropolitan region of Belém. Investigation of the transmission routes in these cases may nevertheless provide important insights into the transmission of HTLV in the region.As HTLV has been shown to spread silently within families, the aim of the present study was to investigate familial aggregations of this infection based on the presence of the virus in at least two relatives of the outpatients. Familial aggregation was demonstrated by the significant difference in infection rates between carrier families  and the relatives tested , indicating that HTLV infection tends to occur in specific members of certain family groups. The seroprevalence rate of 25.6% recorded in family members in markedly higher than the mean rate for Brazilian blood donors in general (0.5%) or in the state of Pará in particular (0.91%) et al. (2001) A high prevalence of HTLV infection in the families of virus carriers has been demonstrated in a number of regions worldwide. In areas of high endemicity in Japan, the prevalence was 38.5% in the family members of infected individuals In a study conducted in the Brazilian state of Minas Gerais, involving family members of 352 HTLV-seropositive blood donors, the prevalence of HTLV-1 was 25.9% Family members who were HTLV-positive were significantly older than those who were seronegative, which is consistent with previous studies, that have shown an increase in infection with age due to the eventual seroconversion of infections acquired early in life or the cumulative risk of infection through the course of an individual's life et al. (2004) Cross-study comparisons of the prevalence rate of HTLV-2 recorded in family members in the present study (26.6%) were not possible due to the lack of data on this virus type. The HTLV-2 virus is endemic in eastern Amazonia, where the highest known rates of infection have been recorded (up to 40%), especially in indigenous communities Little difference was found in the seropositivity rates of the relatives of symptomatic (28.0%) and asymptomatic (23.9%) outpatients, although the combined rate (25.6%) was 28 times higher than that recorded for blood donors from Pará (0.91%). In a Chilean study, an infection rate of 29.1% was recorded in the family members of carriers of TSP/HAM, a rate which was also 30 times higher than that recorded in the general population Positivity rates in mother-child pairs (20.4%) and children breast-fed for more than 6 months (34.0%) were similar to those recorded in previous studies. In Minas Gerais A number of studies have suggested that vertical transmission is more prevalent among women than men The results of the present study revealed much higher rates of transmission to the offspring of HTLV-2 carriers when compared to HTLV-1 carriers (24.1% vs. 5.8%), although there are no data from other studies on which to base comparisons. Analysis of blood samples of native Kayapó from Kararaô village found specific reactivity to HTLV-2 in three of 26 subjects. Two samples were from a mother-child pair. Phylogenetic analysis of this pair showed high similarity (99.9%), thus providing molecular evidence of transmission of subtype HTLV-2c from a mother to her child Horizontal transmission between spouses was significantly more frequent than vertical transmission and was six times more likely to occur from men to women, than from women to men. As a consequence, infection tends to become more predominant in the women members of the family groups over time.There is little consensus on the most common transmission route of HTLV. Vertical transmission was the principal route in a study in Nagasaki, Japan With respect to the transmission of HTLV between spouses, a number of studies have shown that the virus is transmitted more efficiently from men to women. The results of an emblematic study conducted in the 1980s in Japan led to the projection that sexual transmission would be 60 times greater from men to women within 10 years The analysis of risk factors through interviews with patients and relatives does not appear to be effective due to the subjectivity of self-reported data, given that patients may often be unsure of details or have biased memories. Most patients are already of advanced age, for example, and are unlikely to know whether they were breast-fed, and their mothers may have died or be unable to remember which of their children they breast-fed.While the present study provides data on the prevalence of infection among family members, then, and has shown familial aggregations of HTLV infection, these data cannot be used to establish transmission routes reliably. However, further insights may be possible by the sequencing of genes to establish the identity of genomic chains between the members of pairs potentially involved in transmission.Overall, the results of the present study are generally consistent with those published previously. Taken together, these studies indicate emphatically that HTLV is transmitted “silently” within family clusters. The investigation of HTLV infection in the families of virus carriers, whether symptomatic or asymptomatic, is essential in order to limit new infections and minimize the risks of serious diseases such as the two T-cell non-Hodgkin lymphoma cases observed in one of the families monitored in this study.Interruption of the spread of the virus from these clusters will depend on the identification of cases in the population, although in Brazil, infection with HTLV is not treated as a public health problem and has been largely neglected The present study confirms the existence of familial aggregations of HTLV infection in outpatients and, consequently, in the metropolitan region of Belém. Considering family relationships, the importance of sexual contact for the transmission of HTLV in the population, mainly from men to women, was demonstrated. Overall infection rates were similar among family members of carriers of HTLV-1 and HTLV-2. However, the higher rate of infection among children of HTLV-2 carriers compared to those of HTLV-1 carriers suggests an ongoing increase in the prevalence of HTLV-2 in the metropolitan region of Belém. The frequency of serological reactivity among family members increased in proportion to age and was associated with a large proportion of asymptomatic carriers. Overall, the general ignorance of the virus increases the risk of transmission considerably.Checklist S1STROBE Checklist.(DOC)Click here for additional data file.