The activity of ERK2, an essential component of MAP-kinase pathway, is under the strict control of various effector proteins. Despite numerous efforts, no crystal structure of ERK2 complexed with such partners has been obtained so far. PTP-SL is a major regulator of ERK2 activity. To investigate the ERK2–PTP-SL complex we used a combined method based on cross-linking, MALDI-TOF analysis, isothermal titration calorimetry, molecular modeling and docking. Hence, new insights into the stoichiometry, thermodynamics and interacting regions of the complex are obtained and a structural model of ERK2-PTP-SL complex in a state consistent with PTP-SL phosphatase activity is developed incorporating all the experimental constraints available at hand to date. According to this model, part of the N-terminal region of PTP-SL has propensity for intrinsic disorder and becomes structured within the complex with ERK2. The proposed model accounts for the structural basis of several experimental findings such as the complex-dissociating effect of ATP, or PTP-SL blocking effect on the ERK2 export to the nucleus. A general observation emerging from this model is that regions involved in substrate binding in PTP-SL and ERK2, respectively are interacting within the interface of the complex. The MAP Kinase pathway represents one of the most important signaling systems in eukaryotes. It controls a large variety of fundamental cellular processes as proliferation, differentiation, cell survival and apoptosis. Various extracellular stimuli, as growth factors, cytokines, mitogens, environmental stress factors can activate this pathway. Following MAP kinase pathway activation, different cytosolic, membrane-bound or nuclear proteins are expressed, which in turn regulate the transcription of specific genes Kinase Interaction Motif (KIM). This motif is situated between residues 224–239 of PTP-SL and is highly conserved among all members of the PTP-SL sub-family. Within the KIM sequence, there is a PKA consensus phosphorylation motif, Ser 231, which may be phosphorylated and, thus, binding of ERK2 to PTP-SL is impaired PTP-SL is a major regulator of ERK 1/2 MAP kinase activity. There is an unusual reciprocal interaction within the complex formed between these two proteins: ERK 1/2 phosphorylates Thr 253 of PTP-SL and PTP-SL dephosphorylates the regulatory phosphotyrosine (pTyr 185) on the activation loop of ERK 1/2. Dephosphorylation of pTyr 185 causes inactivation of ERK1/2 and its retention in the cytoplasm Crystal structures of inactive and active forms of ERK2 have been reported Despite the relatively numerous reports concerning the docking interaction between MAPKs and specific peptides, there are no structural analyses extended to the interactions of sequences proximal to the KIM region as well as to those involving the kinase active sites. On the other hand it is obvious that the interaction between a MAPK and a protein interactor is not limited to the docking interaction. Specifically, no crystal structure of ERK2 in complex with a substrate or other effector proteins, has been so far reported. Consequently, any finding in this respect would be particularly important in understanding the highly specific nature of ERK2 interactions and the rigorous regulation of its activity. Regarding the ERK2 - PTP-SL complex we previously provided structural support for that the kinase action of ERK2 and the phosphatase activity of PTP-SL involve two distinct conformational arrangements of the interacting partners ab-initio modeling in regions lacking template, followed by simulated annealing optimization. This provides the contact interface in a conformation consistent with PTP-SL phosphatase activity. The final refined version of the model accounts for the structural basis of several reported observations, and also pinpoints novel characteristics of the interaction interface for this physiologically important complex.In this report we investigate the ERK2−PTP-SL complex by isothermal titration calorimetry and chemical cross-linking combined with MALDI-TOF. The resulting experimental data, along with all the rest of structural data existing at hand in the literature are introduced as imposed constraints to build a 3D-model of the ERK2−PTP-SL complex. The model is built starting from the crystal structures of ERK2 and PTP-SL The overall strategy of the experiments was the following: initially, optimal cross-linking reaction conditions for the complex of ERK2 with PTP-SL were established, then the major cross-linked product was isolated, digested with trypsin and the mixture of peptides was analyzed by MALDI-TOF. In parallel, a control experiment with the non-covalent complex (not treated with cross-linker) was subjected to a similar treatment. Identification of mass spectrometric signals corresponding to cross-linking of a peptide from ERK2 with another one from PTP-SL grounded the empirical constraints imposed in the ERK2−PTP-SL model building. A similar strategy was successfully applied for determination of structural models of other protein complexes and multidomain proteins 3) - a homobifunctional agent reacting with amino groups and providing a linker of 12 carbon atoms - and a heterobifunctional agent producing zero length linker - 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (in presence of N-hydroxysuccinimide (NHS)). All cross-linking experiments were performed in absence of ATP in order to favor the conformations of the complex which enable phosphatase activity of PTP-SL on ERK2 and not the kinase activity of ERK2. Various conditions of the cross-linking reaction were tested for both agents, by modifying concentrations of cross-linker, complex concentration, incubation time and temperature. EDC did not yield significant amount of cross-linked product under any tested reaction conditions (data not shown). Instead, BS3 was found to be a relatively efficient cross-linking agent of the ERK2–PTP-SL complex. BS3 concentration and incubation period were the most important parameters for the cross-linking yield and composition of the cross-linked reaction products (data not shown). In all situations, a minor band was observed, corresponding to a cross-linked product of approximately 90 kDa as well as another electrophoretic band, corresponding to the major product, of about 120–160 kDa. To analyze whether the cross-linked products thus obtained are homo- or heteromers, cross-linking reactions were performed in parallel, using either individual ERK2 and PTP-SL proteins or the complex of these two proteins. The complex produced a much higher amount of cross-linked product than the individual proteins (Two common cross-linking agents were tested: bis(sulfosuccinimidyl) suberate  is part of the linker L12 connecting subdomains VIII and IX and is immediately following the P+1 binding area of ERK2 m/z of 2221.75, 2573.88 and 3617.27, there are two alternative assignments and, apparently, this fact generates a large number of possible networks of interacting lysine residues. However, taking into account that only six lysine residues of PTP-SL and three of ERK2 are involved in cross-linking and that a given lysine residue can only be involved in a single cross-linking interaction, it can be inferred that only three alternatives are possible as concerning the networks of the cross-linked lysine residues  by ERK2 (75 µM). The isotherm could be best fitted to a single-site binding model, resulting in a stoichiometry of 1∶1 (n = 1.06±0.005), an affinity of Ka = 6.6±0.5×106 M−1 (Kd = 0.15 µM) and a reaction enthalpy ΔH = −21.0±0.1 kcal/mol. From the experimentally determined changes in enthalpy and free energy upon the protein/protein interaction, one can calculate the entropy contribution, TΔS = −11.5 kcal/mol. This negative value means that the entropy term is unfavourable to the interaction, likely due to a decreased conformational freedom of the two proteins upon binding. The intermolecular interaction is therefore driven by the enthalpic contribution. Similar results were obtained when the wild type PTP-SL was used in the ITC experiments.In order to get additional elements concerning the stoichiometry of the complex, isothermal titration calorimetric (ITC) measurements were also performed. ab-initio modeling subjected to all the existing experimental constraints known to date, including those derived from cross-linking experiments  of PTP-SL and (aa 6–358) of ERK2 was generated, starting from the existing crystal structures . This was named PECr. In the second step the PECr model was used to build the rest of the PTP-SL cytoplasmic domain (aa147–253). This was performed by eriments .PTP-SL-pY185ERK2, P445PTP-SL -pY185ERK2 and D446PTP-SL -pY185ERK-2). Rosetta Dock has the advantage of using explicit flexibility of the side chains and was shown to be one of the best ranked protein-protein docking methods, according to the CAPRI evaluation For building the PECr model, we proceeded as follows. The PTP-1B complexed with a hexapeptide substrate containing phosphotyrosine (PDB code 1PTU), was used as template. On this template, both PTP-SL (aa 254–549) and the ‘new’ substrate, the phosphorylated ERK2, were superimposed, in order to orient the ERK2 in the phosphorylation region similarly to the hexapeptide substrate in the template. This gives the triangulation of ERK2 with respect to PTP-SL. In order to find the best surface of complementarity of the two protein surfaces and to eliminate all steric conflicts, this structure was used as the starting point for docking optimization using Rosetta Dock with constraints imposed on the distances between the WPD loop of PTP-SL and phosphotyrosine on the lip of ERK2 (residues W444It is worth noting here that the PTP-1B-hexapeptide complex (1PTU) was a better template for PECr model than the CDK2-KAP complex ab-initio techniques will yield only a low-resolution structure of rms >5 Å as any prediction for proteins lacking homologues ab-initio procedures and to be connected to the PTP-SL (aa224–549) model, both under the docking constraints imposed by the PECr model surface and distance constraints derived from cross-linking experiments and polypeptide connection to the rest of the model. As multiple paths are possible, a repertoire of over 100 ab-initio structures was generated with Rosetta. These 3D structures were mapping slightly different PTP-SL stretches of 78–90 amino acids comprising the aa147–223 region, in order to maximize the 3D structural diversity of the ab-initio models. Structures were clustered according to their rms deviation and representatives of the top 5 largest clusters were used for docking with constraints imposed by the PEC model surface, the connection at the PTP-SL aa223 end, and the results of cross-linking experiments. From these, the best-matching cross-linking constraints and PEC surface complementarity, was used to complete the overall model of PTP-SL (aa147–549). This final version of PTP-SL (147–549)-ERK2 docking model, named PECf was kept for further structure quality assessment, representation and analysis (The PECr model was then used as a starting point to generate the missing region of the cytosolic part of PTP-SL (aa147–253), by combined docking, ab-initio and homology modeling. At present, there are no structural data on this region, as crystallization attempts of the complex have failed so far. Interestingly, this is consistent with predictions resulting from profiling the intrinsic disorder propensity of PTP-SL by two distinct methods , based eanalysis . From vaIntermolecular interaction energy analysis at the interface between ERK2 and the low resolution model of the disordered (aa147–253) region of PTP-SL, suggests that the top contribution to the desolvation component of free energy is provided by PTP-SL residues: V182, I205, T209, M220, L225, L235, L237 and M239, while the top contribution to the electrostatic component is provided also by PTP-SL: K155, V190, Q198, R228, R229, E246. Particularly the KIM region seems to contribute significantly to ERK2-PTP-SL complexation in this area of interface.The PECf model shows that the residues L235, L237, M239 in PTP-SL conserved among the KIM containing D-motif docking peptides, are in van der Waals contact with L113, F127 and L155 residues from ERK2 and form a hydrophobic cluster. This cluster is located near the KIM motif salt bridges, thus making the whole region a strong stabilizing patch of the complex . We analPTP-SL is situated on the solvent exposed area of PTP-SL within the KIM region. Although it is not oriented to the interface it is possible that S231PTP-SL phosphorylation will re-orient the side-chain during conformational changes, thus impairing PTP-SL interaction with ERK2 According to the model the PKA phosphorylation site S231PTP-SL and S282PTP-SL situated on the loop linking α1′ and α2′ helices form hydrogen bonds with S264ERK2 situated on the loop α2L14, apparently involved in substrate recognition ERK2 is relatively conserved among those MAP kinases where the similar loop α2L14 is present, the tandem serine residues S281–S282PTP-SL are specific for PTP-SL but not for other KIM-containing PTPases. Consequently, this interaction seems to contribute to the mentioned specificity of PTP-SL interaction with ERK2.Our results also indicate that the KIS sequence - immediately following the KIM region and contributing together with KIM to differential binding of PTP-SL to ERK1/2 and p38 - is also involved in specific interactions with ERK2. For instance, residues S281PTP-SL - part of α2′ – interacts with H230ERK2 from L13 through stacking forces and hydrogen bond involving peptide bond of F289PTP-SL. The significance of H230ERK2 located on L13 resides in its “strategic” position, right before helix G which defines together with helix D, the substrate binding groove of ERK2 ERK2, P227ERK2, K229 ERK2 and Y231 ERK2 also take part in binding PTP-SL at the interface with ERK2. Except for the conserved amino acid P224, the other residues which belong to this loop seem to be rather specific for ERK2 but not for other MAP kinases. On the other hand, the interacting partner residue F289PTP-SL is highly conserved. Experimental findings support the fact that H230ERK2 plays a role in substrate binding by ERK2. Thus, H230N mutation, suggested to be a gain-of-function mutation for ERK2 Helix α2′ of PTP-SL also plays an important role in interacting ERK2. Thus, F289ERK2 and W190ERK2 and termed “P+1” specificity region, represent such an example. One of the most important of these residues is phosphotyrosine pY185ERK2, located on the phosphorylation lip of ERK2. According to our model, its binding to the active site of PTP-SL is assisted by hydrophobic (stacking) interactions with the highly conserved residue Y313PTP-SL which in turn is stabilized through hydrophobic forces between its aromatic ring and the aliphatic portion of the side chain of R312PTP-SL. This is an interesting finding, because it confirms previous structural results obtained for the complex of PTP-1B with peptide substrates. Thus, Y46 of PTP1B  was found as part of the substrate phosphotyrosine recognition loop PTP1B and of the general aspartic acid of the catalytic WPD loop in the catalytically inactive domain D2, confers robust activity in several PTPs PTP-SL) is considered a putative substrate binding site PTP1B and Y46PTP1B are conserved residues and belong to the NXXKNRY motif, one of the ten conserved motifs which defines the PTP family The model shows that other structural regions of ERK2 responsible for substrate binding, are involved in interface interactions as well. Residues of the loop L12 of ERK2, encompassed between Y185The orientation of a phosphorylated hexapeptide interacting with PTP-SL (1PTU) was used as a key constraint in orienting ERK2 with respect to PTP-SL. Thus, PECf conformation does model the complex during ERK2 dephosphorylation. As phosphorylation and dephosphorylation require different conformations Results of the cross-linking experiments, combined with SDS-PAGE and MALDI-TOF analysis of the cross-linked products, as well as the results of gel-filtration experiments on PTP-SL and its complex with ERK2, support the idea that the complex is a mixture of heterodimer and heterotrimer (composed of two PTP-SL and one ERK-2 molecule). By contrast, according to the isothermal calorimetric titration, the stoichiometry of the complex is one PTP-SL to one ERK2 molecule. A possible explanation of these contradictory results might be that the concentration of the complex used in cross-linking and gel-filtration experiments was much higher due to technical constraints, than that used in the ITC experiment. Thus, it may be assumed that in fact an equilibrium between 1∶1 heterodimer and 2∶1 heterotrimer forms exists and high protein concentration shifts the equilibrium towards the formation of heterotrimeric form.in vivo conditions, the complex is in fact a larger entity. Indeed, it has been shown that a member of the PTPBS family of PTPs (human homologues of PTP-SL) forms, with oxysterol-binding protein (OSBP), serine/threonine phosphatase PP2A and cholesterol, a cca. 440 kDa oligomer which regulates phosphorylation of ERK2 in HeLa cells The sub-micromolar range of the dissociation constant of the heterodimer as obtained by calorimetric measurements (0.15 µM) provides a quantitative support for the previously reported experimental finding that this complex involves a very tight interaction between the two proteinsPTP-SL and Y313PTP-SL from the KNRY substrate recognition loop of PTP-SL, instead of P254PTP-SL. Therefore, the phosphorylating activity of ERK2 can be performed only by a rearrangement of the conformation adopted by the complex, in which T253PTP-SL is directed to the active site of ERK2 and consequently, binding of the neighbor residue P254PTP-SL by the “P+1” specificity region on the L12 loop becomes possible. Given that the presence of the KIM region of PTP-SL is essential for complexing ERK2 An interesting principle which seems to govern the interactions between PTP-SL and ERK2, is that regions responsible for substrate binding in individual proteins are reciprocally interacting within the complex, as illustrated by the PECf model. Thus, the KIM, KIS and NXXKNRY motifs from PTP-SL interact with L16, α2L14 and L12 loops of ERK2, respectively, all involved in binding of specific substrates. As concerning the interaction of the L12 loop from ERK2 with KNRY motif of PTP-SL it confirms a former hypothesis that the ERK2 catalyzed phosphorylation and the PTP-SL catalyzed dephosphorylation require different conformations of the complex An important feature of the complex formation between PTP-SL and ERK2 resides in the fact that PTP-SL blocks ERK2 translocation to the nucleus, due to its KIM-dependent binding of ERK2 PTPRR is a human homologue of PTP-SL and there are reports indicating its involvement in nerve growth factor signaling, thus representing potential target in the therapy of neurodegenerative processes, like Alzheimer disease The cDNA corresponding to the full cytoplasmic region of PTP-SL (aa147–549) and its abridged form (aa212–549) were inserted into the pGEX-4T1 expression vector  and the resulting recombinant plasmids were subsequently used to transform DH5α host cells. cDNA of ERK2 was cloned into the pHAT2 expression vector 3 was obtained from Pierce . The PTP-SL- ERK2 complex, in 100 mM HEPES buffer, pH = 7.5 with 300 mM NaCl, was incubated with freshly prepared BS3 solution. This solution contains BS3 dissolved in 5 mM citrate buffer, pH = 5. Reaction conditions were varied in the following ranges: 0.1–1 mM cross-linking reagent, 2.2–5.54 µg/µl complex, 30–120 min. reaction time and temperature between 0°C and 25°C. The cross-linking reaction was terminated by the addition of 1/6 volume of 300 mM Tris buffer, pH = 7.5. The cross-linking reactions of PTP-SL and ERK2 with BS3 were performed under the same optimal conditions as in the case of the complex.BSGel electrophoretic separation of cross-linked products and non-cross-linked molecules was performed using 12% polyacrylamide gels under denaturating conditions To purify the cross-linking conjugate gel filtration was carried out at room temperature with a Superdex 200 HR column 2.2/58. The column was equilibrated with gel filtration buffer: 10 mM Hepes, 150 mM NaCl, 0.1 mM EDTA, 5 mM DTT, 1% SDS, pH = 7.5. A volume of 200 µl cross-linking mixture was incubated for 5 min with 40 µl gel filtration buffer at room temperature, then boiled for 5 min. The boiled sample was centrifuged at 14,000 rpm, 10 min. 220 µl of mixture was applied on the column. Fractions of 500 µl were collected at a flow rate of 0.4 ml/min. A Superdex 200 HR 10/30 analytical column was used in the size exclusion chromatography experiments, to calculate MWs of the PTP-SL forms: 212–549, 147–549, 147–549(C480S) alone and of the various forms of their complexes with ERK2. The gel filtration buffer was the same as previously mentioned but without SDS. For each run 100 µl sample was applied on the column. The flow rate was established at 0.5 ml/min. Gel Filtration Standard (BioRad) was used for MW calibration.a) and enthalpy of complex formation (ΔH). The experimental data allow calculation of the free energy change (ΔG) and of the entropy term (TΔS), according to the classical thermodynamic formulae: ΔG = −RT×ln Ka and ΔG = ΔH−TΔS. All the experiments were repeated twice, and give similar results. The control experiment, consisting in injecting an ERK2 solution into the buffer, shows that the heat of dilution is negligible. The raw calorimetric data were fitted to a simple model with a single type binding site. Three parameters are thus optimized by minimizing the mean square distance between experimental and calculated points. For the stoichiometry n, the optimized value was n = 1.06 with the standard error 0.005, reflecting a great confidence for the 1∶1 stoichiometry. In fact, for affinity values larger than 106 M−1 the isothermal binding curve has a clear sigmoidal shape with the inflection point indicating the stoichiometry value.Thermodynamic parameters of molecular interactions between ERK2 and PTP-SL at 30°C were investigated by ITC using a MicroCal MCS instrument . A PTP-SL solution (8–10 µM) in the calorimeter cell (1.337 mL) was titrated by ERK2 (75–100 µM) using automatic injections of 8–10 µL. The first injection of 2 µl was ignored in the final data analysis. Integration of the peaks corresponding to each injection and correction for the baseline were done using Origin-based software provided by the manufacturer. Fitting of the data to an interaction model results in the stoichiometry (n), equilibrium binding constant , mixed with digested samples and applied to a stainless-steel probe plate. Spectra were acquired with a Voyager DE-PRO workstation, using a delayed extraction, linear mode. Mass accuracies were obtained with external calibration. All reported masses are averaged values. The purified, cross-linked complex was also directly analyzed by MALDI-TOF, using sinapinic acid as matrix. The MALDI-TOF spectral data corresponding to the tryptic peptide mixture were introduced in Search-X-links program The cross-linked complex purified by size exclusion chromatography (pooled fractions corresponding to 120–160 kDa) was first precipitated with methanol/chloroform according to a reported procedure Homology modeling was performed with Insight II software package from Accelrys. The Homology module was used for coordinate transfer and loop generation. Local simulated annealing and energy minimization during modeling steps were performed via the Discover module with cvff force field. - Instrinsic disorder assignment was performed with DISOPRED Ab initio modeling was performed using HMMSTR/Rosetta Docking was performed with InsightII, RosettaDock 2 were imposed on α-carbons of aminoacids along the polypeptide chain found in definite secondary structure states . Simulated annealing was followed by extensive constraint-free energy minimization. - In both MD-SA steps and model stability tests MD simulation was carried with CHARMm using CHARMM27 force field 3 box, at constant temperature (NVT dynamics) at 300 K and RMSD was measured with VMD. Intermolecular free energy calculations were performed with FastContact


The proportion of older people is increasing worldwide. Globally, it is estimated that older people (those 60 years or older) constitute more than 11% of the population. As the HIV/AIDS pandemic rages in sub-Saharan Africa (SSA), its impact on older people needs closer attention given the increased economic and social roles older people have taken on as a result of increased mortality among adults in the productive age groups. Few studies have looked at older people and their health in SSA or indeed the impact of HIV/AIDS on their health. This study aims to assess the effect of being directly or indirectly affected by HIV/AIDS on the health of older people in two Nairobi slums.st October 2006. Health status was assessed using the short SAGE  form and two outcome measures – self-rated health and a composite health score – were generated. To assess HIV/AIDS affected status, respondents were asked: Have you personally been affected by HIV/AIDS? If yes, a follow up question: "How have you been personally affected by HIV/AIDS?" was asked. Ordinallogistic regression was used in models with self-rated health and linear regression in models with the health score.Data were collected from residents of the Nairobi Urban Health and Demographic Surveillance area aged 50 years and above on 1About 18% of respondents reported being affected by HIV/AIDS in at least one way, although less than 1% reported being infected with HIV. Nearly 60% of respondents reported being in good health, 27% in fair health and 14% in poor health. The overall mean health score was 70.6 (SD: 13.9) with females reporting worse health outcomes than males.Respondents directly or indirectly affected by HIV/AIDS reported worse health outcomes than those not affected: mean health score: 68.5 and 71.1 respectively , and an adjusted odds ratio of reporting poor health of 1.42 (95%CI: 1.12–1.80).Poor health outcomes among older people affected by HIV/AIDS highlight the need for policies that target them in the fight against HIV/AIDS if they are to play their envisaged care giving and other traditional roles. The proportion of older people is increasing worldwide. It is estimated that older people (those 60 years or older) constitute more than 11% of the population globally, over 20% of the population in developed nations and about 8% in developing ones. The proportion of older people in developing countries is expected to rise to about 20% by 2050 . . 3]. MorThe proportion of HIV-infected older people has increased in recent years in developed countries where use of antiretroviral therapy is widespread -11 but tThe impact of the HIV/AIDS pandemic on other age groups such as young children, adolescents and adults in reproductive age has been extensively studied ,17. TherThis study therefore aims to fill existing knowledge gaps on the impact of HIV/AIDS on the health of older people. It assesses the direct and indirect effects of HIV/AIDS on the health of older people in two Nairobi slums in a quantitative manner using standard measures of health and considering other dimensions in which older people can be affected by HIV/AIDS.2 respectively. The informal, and hence non-permanent, nature of the slums settlements encourages the official neglect of these communities in the provision of infrastructure and social services. There are only three public health facilities serving the two communities, but these are located on the outskirts of the slums, serve big catchment areas, are closed at night, and often lack drugs and supplies. About three quarters of the residents buy water from vendors and only 22% have access to piped water compared to 78% in the rest of Nairobi. Only 7% of residents have a flush toilet, 84% use pit latrines and the rest have no toilet facility [Data were collected from all older people residing in the two slum communities where the African Population and Health Research Center (APHRC) is implementing the longitudinal Nairobi Urban Health and Demographic Surveillance System (NUHDSS). The NUHDSS covers the two slums of Korogocho and Viwandani in Nairobi, Kenya's capital and commercial centre. The two slums are located about 5–10 km from the city centre and occupy an area of 0.45 and 0.52 kmfacility  HIV/AIDSfacility , and clofacility .st October 2006) were identified as ever being resident in the surveillance areas from the most up-to-date NUHDSS database. Out of these, complete information was collected from 2078 respondents, who were found in their homes after repeated visits and who consented to participate in the study; 102 people refused to participate.The NUHDSS has been operational since January 2003 and data on core demographic events  are collected from members of about 22,000 households in the defined geographic areas, and updated every four months during routine Demographic Surveillance System (DSS) rounds. A total of 2696 older people ? A follow up question: "How have you been personally affected by HIV/AIDS?" with multiple response options was then asked to those who answered "yes" to the first one. Responses included: i) currently caring or cared for someone infected with HIV/AIDS, ii) currently caring for orphaned children, iii) loss of support from adult children who died of, or currently sick with, AIDS, iv) self-reported HIV/AIDS infection, v) loss/reduction of community support to older people as a result of the HIV/AIDS epidemic, vi) loss of spouse to AIDS, vii) any other way. The above response categories were based on findings from a qualitative study in which older people in the same study area described the ways in which they had been affected by HIV/AIDS.To assess whether one was HIV/AIDS affected, respondents were asked: not included in the category of being unaffected (x = 0). This procedure was used in defining all the other exposure variables.From the assessment of being HIV-affected, we derived binary exposure variables for the different ways in which older people could be affected by HIV/AIDS. To derive exposure status, respondents were first grouped into two broad categories of those affected and those not affected at all by HIV/AIDS. The former were then re-categorised according to the way in which they were affected. Respondents affected by HIV/AIDS in ways other than the one under investigation in individual models were not included in the specific analyses. For instance, for the exposure variable "HIV-infected" (x = 1), those who were affected by providing care to HIV/AIDS infected individuals or by providing care and support to orphans or those who had lost support from family and community were Health status was assessed using the short form of the individual SAGE  questionnaire . This foIn addition, the SAGE form has questions on functioning assessment using items in the Activities of Daily Living/Instrumental Activities of Daily Living (ADL/IADL) tool as well as on subjective wellbeing and quality of life.For purposes of this analysis we used two measures of health status:In general, how would you rate your health today, would you say your health is Very good (1), Good (2), Fair (3), Bad (4), or Very bad (5)? A three-category outcome variable for Good (very good and good), Fair (fair) and Poor (very bad and bad) health was derived by collapsing the categories in parentheses.i) Self-rated health status which is derived from the question: ®, a Rasch measurement software package . IRT uses Maximum Likelihood Estimation methods to model the relationship between a person's health status and their probability of responding to each question in a multi-item scale. Typically, questions ask about how much difficulty the respondent had had in the preceding 30 days with tasks or activities in the eight domains. Responses range from no difficulty to extreme difficulty on a five-item scale. An example of a question in the mobility domain is: "Overall in the last 30 days how much difficulty did you have with moving around?" for which the response items were: 1(None), 2(Mild), 3(Moderate), 4(Severe), 5(Extreme/cannot do). Each item is modeled to have a set of parameters which describe the relationship between the item and the measured construct as well as how the item functions within a population. The health score is then transformed to a scale of 0 to 100 . More details on the application of the IRT approach to computing patient-reported health outcomes are available in the paper by Chang and Reeve [ii) Composite health scores derived using item response theory (IRT) parameter estimates in Winstepsnd Reeve . Health For the first measure of health, ordinal logistic regressions were carried out using HIV-affected status and the different ways of being affected as the exposure variables controlling for sex, age , marital status , education level , wealth index (quintiles), ethnicity , and slum of residence.® Version 9.For the second measure of health, linear regression was performed using the composite health score as the outcome variable. The regression model also included the covariates listed above in the same format. In both analyses, interaction terms between sex and the main explanatory variables were tested and none were found significant. All analyses were done in STATAThere are many more males than females in the study population and the sex ratio is 1.00 to 0.56. More than two thirds of males are in the 50–59 age group compared to a little over half of females in the same age group. There are also more females in the 70+ age groups compared to males. Overall, 68.0% of respondents are married or have a partner though there are large sex differences with only 30.3% of females being in partnership compared to 89.1% of males. The majority of females are formerly married  with widows accounting for more than two fifths of the entire sample of females. Almost half of the females have no formal education compared to less than 20% of males. Females are more likely to report being affected by HIV/AIDS than males. More details can be found in the descriptive characteristics table in additional file In all ways in which one can be HIV-affected, females were twice as likely to be affected as males except for perceived loss of community support. Overall <1% of respondents reported being HIV infected, 7.7% had cared for or were currently caring for a family member with AIDS, 5.3% had cared for or were currently caring for orphans, 1.8% had lost social and financial support due to the illness and/or death of their adult children, 4.0% had lost general community support normally given to older people and 0.3% had lost a spouse to HIV/AIDS. About 5% of respondents were affected in more than one way and in total 17.8% were affected by HIV/AIDS in at least one way.Overall, 59.7% of respondents reported having good health, 26.6% reported fair health and 13.7% reported poor health . There were noticeable differences between those affected by HIV and those not affected in these proportions. The respective percentages were 54.6, 28.9 and 16.5 for the HIV affected and 60.8, 26.1 and 13.1 for those not affected. The distribution of older people who reported bad or very bad health in different categories of key covariates is shown, in the descriptive characteristics table in additional file The overall mean health score was 70.6 SD 13.9) and the median score was 67.5. For females the mean score (SD) and median score were respectively 66.1 (12.4) and 64.2, while for males they were 73.1 (14.1) and 69.5. HIV/AIDS affected respondents had slightly lower health scores (mean 68.5) than those unaffected . Respondents who were HIV infected had lower health scores than those not affected by HIV in any way (66.8 vs. 71.1) though the difference did not reach statistical significance. In the same way, those who had been affected through care giving for HIV/AIDS family members, care giving for orphans, loss of support through death of offspring, loss of community support, had significantly lower health scores than those who had not been affected in any way. The respective scores were 66.3 , 67.3 , 66.0  and 65.6 . The mean health scores for categories of key covariates are shown, in the table in additional file 3.9 and tIn unadjusted ordinal logistic regression models, HIV-affected respondents had higher odds of being in the poor health category than those not affected in any way with an overall odds ratio of 1.29 (95% CI: 1.04–1.61). Detailed results are shown in Table Respondents affected by HIV in all ways, except loss of support from deceased offspring and relatives, showed higher odds of being in the poor health category in unadjusted models. Only the odds ratio for those who were affected through care for HIV/AIDS affected family members reached statistical significance. In adjusted models, these results were only slightly changed. The odds ratios for being HIV/AIDS affected in any way and for care giving for ill family members were enhanced in adjusted models and retained statistical significance.In all the adjusted ordinal logistic regression models, sex, age and slum of residence had significant effects on self-reported health. Females were more likely to report being in poor health than males, there was a positive age gradient whereby older respondents were more likely to report being in poor health than younger ones and residents of Korogocho were more likely to report being in poor health than those of Viwandani.In unadjusted linear regression models, respondents who were HIV-affected in any way had lower mean health scores than those who were not, as shown by the negative β-coefficients Table . Scores In adjusted linear regression models, regression coefficients were mostly enhanced and hence health scores were lower than in unadjusted models. Significantly lower health scores were observed among those affected by HIV/AIDS in any way and those affected in all specific ways except those with self-reported HIV infection and perceived loss of community support.In all the linear regression models, sex, age, slum of residence, education level, and wealth index had significant effects on the health score. The direction and magnitude of the effects were similar to those in the ordinal logistic regression models above for the first three covariates. There was an education gradient whereby those with more than six years of formal education had the highest health scores and those with no formal education the least. For wealth index, a non-monotonic relationship was observed whereby respondents in the first and fourth quintiles had the lowest health scores and those in the second had the highest.This study aimed to assess the effect of HIV/AIDS on the health of older people in two Nairobi slums. More than one sixth of respondents aged 50 and over, were affected by HIV/AIDS in one way or the other mostly through care giving for HIV/AIDS-infected family members and HIV/AIDS orphaned children. About 14% of all respondents reported bad or very bad self-rated health with about 17% of the HIV-affected respondents reporting the same. There were significant differences in the mean health scores of those affected by HIV and those not affected, suggesting that HIV-affected older people have worse health outcomes than those not affected.The sex and age distribution of the study population is atypical and is unlike what one would expect in the general population. It however reflects the nature of the two informal settlements which are predominantly occupied by labor migrants, most of whom are young males. In all age groups in the NUHDSS, after the age of 25 years, there are almost twice as many males as females migrating to or residing in the two informal settlements and this is also true for the 50+ age group. The high proportion of formerly married females reflects the underlying reasons for female migration into the slums which include marital strife. In addition, while most males remarry after the death of their spouse, few females do so in this setting, especially at older ages.There are few studies that have assessed the health and wellbeing of older people in an African setting despite the increasing realization that population aging in the continent is a key demographic trend that will have significant impact on social support and fledgling health systems ,20. Few Generally, the absence of studies in similar settings makes it hard to interpret some of the findings. For instance, it is difficult to judge whether 13.7% – the proportion of respondents who reported having bad or very bad health – is high or low or is as expected. In addition, the absence of data on a comparison population and an assessment of contextual factors (if such a population existed) make it difficult to interpret the mean composite health score of 70.6 (we) observed in this study population. However, differences observed between older and younger respondents, between males and females as well as the expected gradient with educational status point to a high degree of internal validity. Gender differences in health status have been observed in studies in other settings where females fare worse than males -31 and sHIV-affected respondents exhibited lower mean health scores and had higher odds of being in the poor health category. However, the small number of respondents in the different exposure categories led to lack of significance in most estimates in the ordinal logistic regression models.The finding that older people in care giving roles for HIV-infected family members have worse health outcomes than those not affected by HIV in any way is unsurprising. This could be explained by the relatively higher physical and emotional demands on care givers for terminally ill family members as has been found in other studies ,18,20. CSome older people reported they had been affected by HIV/AIDS through loss of community support and neglect of the day-to-day needs of older people. Findings from other studies in this community suggest that community support is largely limited to major needs such as funerals and hospitalization but not for what may often be considered routine needs such as food or minor illnesses . While tThe proportion of respondents that were or had been involved in care giving roles as well as the proportion of older people reporting being affected by HIV through loss of support from deceased or ill family members are surprisingly low given the high burden of HIV/AIDS in the community . HoweverThe self-reported HIV prevalence of 0.8% in this population is very low and is most probably an underestimate given the fact that the HIV prevalence in the adult population in the two slums (11.5%)  is much Self-reported health status, though widely used in other settings, has not been as widely used in African settings and its validity as a measure of health has not been established. It is known that the validity of self-reported measures of health and their reliability are influenced by underlying socio-cultural factors including basic and health literacy, cultural perceptions of illness, disability and health among others ,34. The The use of a composite measure of health masks difficulties with activities in individual domains of health or domain-specific health outcomes. For instance, the study did not investigate the extent to which variations in mean health scores could be dependent on particularly large scores in domains such as pain and discomfort, affect and sleep which are likely to be more affected if one is HIV-affected compared to domains like cognition, vision and breathing. Given the large number of domains involved (eight), it would have been impossible to do justice to all of them in one paper and show how, difficulty in each domain, could be explained by the various ways of being HIV-affected. The sample size is also too small for such detailed analyses.This being a cross-sectional study, temporality cannot be established. It is possible that there is a selection effect, for example, among respondents who cared for HIV/AIDS infected relatives or those who cared for orphans. Older people in poor health would not be expected to care for relatives or orphans unless no other relative was available. Longitudinal studies that establish health state dynamics as a function of being HIV-affected taking selection effects into account are recommended.The importance of older people in the fight against HIV/AIDS cannot be overemphasized. Our study, the first of its kind in the African region aimed to demonstrate the effect of being HIV-affected on the health of older people. The worse health outcomes exhibited by older people affected by HIV/AIDS highlight the need for policy makers, development partners and civil society organizations involved in HIV/AIDS initiatives to develop policies and programs that specifically target older people. Such programs should include those that improve HIV/AIDS and other general health services for older people and those that increase uptake for counseling and testing services. Older people should also be targeted by initiatives aimed at mitigating the social and economic impact of HIV/AIDS on vulnerable groups.The authors declare that they have no competing interests.CK contributed to the design of the study instrument, did the data analysis and literature review, wrote the first draft and contributed to the interpretation of the results, ACE contributed to the conceptualization of the study, writing the paper and interpretation of the findings, EZ conceptualized the study, contributed to the writing of the paper and interpretation of the findings, JF contributed to the overall conceptualization of the study and the design of the survey instrument, contributed to writing the paper and interpretation of the findings. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Descriptive characteristics table. Descriptive characteristics of the study population of older people in the Nairobi DSS, November 2006 to February 2007Click here for file


Negative public reactions to emerging infectious diseases can adversely affect population health. We assessed whether social support was associated with knowledge of, worry about, and attitudes towards AIDS and severe acute respiratory syndrome. Our findings suggest that social support may be central to our understanding of public responses to emerging infectious diseases. The ability of public health institutions to effectively manage emerging infectious diseases (EIDs) and mitigate their consequences is partly a function of public reaction to an epidemic. Negative reactions may vary from denial to panic to stigmatization. Denial or minimization of the threat of an EID by the population at risk can impede prevention efforts and increase transmission. Alternatively, an overreaction to the threat of an EID can overwhelm public health systems and resources, divert resources from effective disease control activities, and lead to severe economic losses in areas affected by the disease. Stigmatization can increase problems of persons with the disease and facilitate transmission because persons with or at risk for the disease may avoid seeking healthcare and because governments may attempt to suppress information about EIDs, considering their potentially severe economic consequences. From a public health policy perspective, identifying personal characteristics and resources that predict responses of persons to EIDs is important to improve the ability of the public to learn about, rationally appraise the threat of, and minimize stigmatization of EIDs.Determinants of a person’s knowledge, worry, and stigma about EIDs are poorly understood. Although the quality of information conveyed to the public by various sources  and epidemiologic characteristics of the disease  are important, early empirical work suggests that responses to EIDs are influenced by characteristics that include a greater stock of personal resources. We recently reported that less education was associated with being poorly informed about severe acute respiratory syndrome (SARS), very worried about AIDS, and having more stigmatizing attitudes toward AIDS and SARS (In this study, we assessed whether social support was independently associated with knowledge of, worry about, and attitudes toward AIDS and SARS in a representative sample of persons living in the New York City metropolitan area. Social support is defined as the functional aspect of social relationships (>18 years of age who were living in the New York City metropolitan area on September 11, 2001. The cohort was recruited through a random digit dial telephone survey from March 25 through June 25, 2002. Contact information was obtained and follow-up interviews were conducted from September 25, 2002, through January 31, 2003, and from September 24, 2003, through February 29, 2004. Data regarding knowledge of, worry about, and attitudes toward AIDS and SARS were collected among a randomly selected subset of participants in the second cohort follow-up. The total sample included in this analysis, for whom data about AIDS, SARS, and social support were incomplete, consisted of 914 persons; response rate for eligible participants was 56%. Each participant received a $10 incentive. Additional details on sampling are provided elsewhere (Data for this study came from a cohort of persons We collected information about respondent sex, race/ethnicity, age, educational attainment, marital status, and household income at baseline. Social support was assessed by using a 5-item modified version of the Medical Outcomes Study social support scale . FurtherWe described sociodemographic characteristics and level of social support of the respondents and created multivariable models predicting knowledge of, worry about, and stigmatizing attitudes toward AIDS and SARS, including covariates thought to potentially confound the relationship between social support and each outcome. Analyses were weighted to correct potential selection bias related to the number of household telephones, persons in the household, and oversampling, as well as to make the sample demographically similar to the New York City metropolitan area population according to US Census 2000 estimates. We used SUDAAN software to estimate standard errors and adjust analyses for the weighting (Of the 914 participants, 45.4% were male, 54.1% white, 7.6% Asian or other race, 18.5% black, and 19.8% Hispanic . In multReporting higher levels of social support was independently associated with greater knowledge of AIDS but not of SARS. Lower levels of social support were associated with more stigmatizing attitudes toward SARS but not AIDS. These patterns may be explained by differences in the epidemiology of AIDS and SARS in New York City. AIDS has been associated with tremendous illness and mortality rates in New York City (Social support was inversely associated with worry about both AIDS and SARS, which suggested that association between social support and AIDS and SARS my persist despite differences in the epidemiology of EIDs. These findings are consistent with studies showing that social interactions are associated with perceptions of the risk for HIV/AIDS (This study has several limitations. First, covariates were treated as time-fixed, which may have resulted in residual confounding. Second, social support is a multidimensional construct; specific dimensions may differentially influence knowledge of, worry about, and stigmatizing attitudes toward EIDs. Finally, for a cross-sectional analysis, it is difficult to establish temporality between levels of social support and knowledge of, worry about, and attitudes toward AIDS and SARS. Observed associations between social support and responses to EIDs may be explained by a common unmeasured covariate.Emerging and reemerging infectious diseases will continue to profoundly affect human health. The ability to effectively mitigate disease consequences will depend, in part, on minimizing negative public responses. We showed that social support may be central to public responses to EIDs. Further research investigating the pathways linking social support to responses to EIDs may inform interventions that help guide public health and official responses to these diseases.Variables used in multivariable models to assess the relationship between social support and knowledge, worry, and stigmatization of AIDS and SARS in 914 study participants, New York, New York, metropolitan area*


This study uses surveillance, survey and program data to estimate past trends and current levels of HIV in Botswana and the effects of treatment and prevention programs.Data from sentinel surveillance at antenatal clinics and a national population survey were used to estimate the trend of adult HIV prevalence from 1980 to 2007. Using the prevalence trend we estimated the number of new adult infections, the transmission from mothers to children, the need for treatment and the effects of antiretroviral therapy (ART) and adult and child deaths. Prevalence has declined slowly in urban areas since 2000 and has remained stable in rural areas. National prevalence is estimated at 26% (25–27%) in 2007. About 330,000  people are infected with HIV including 20,000 children. The number of new adult infections has been stable for several years at about 20,000 annually . The number of new child infections has declined from 4600 in 1999 to about 890 (810–980) today due to nearly complete coverage of an effective program to prevent mother-to-child transmission (PMTCT). The annual number of adult deaths has declined from a peak of over 15,500 in 2003 to under 7400  today due to coverage of ART that reaches over 80% in need. The need for ART will increase by 60% by 2016.Botswana's PMTCT and treatment programs have achieved significant results in preventing new child infections and deaths among adults and children. The number of new adult infections continues at a high level. More effective prevention efforts are urgently needed. Botswana has one of the highest levels of HIV in the world. UNAIDS estimated that adult prevalence was about 24% in 2005, higher than any other country except Swaziland Comprehensive assessments of the demographic impact of the epidemic were conducted for Botswana in 2000 The HIV surveillance program conducts annual HIV surveys among women aged 15–49 years attending ante-natal clinics. Surveillance has been conducted annually since 1992 and now includes 24 sites The Botswana AIDS Impact Survey II (BAIS II) conducted in 2004 measured the prevalence of HIV infection in the population aged 18 months and above The UNAIDS Reference Group on Estimates, Models and Projections has developed several tools to estimate national prevalence. One of these tools, the Estimation and Projection Package (EPP), is used in most countries in sub-Saharan Africa to estimate prevalence trends from surveillance and survey data in countries with generalized epidemics The epidemic model uses four parameters to determine the prevalence trend over time: the start year of the epidemic, the initial force of infection, the proportion of the population at risk of infection and the rate of replenishment of the population at risk when it is depleted by AIDS deaths. EPP generates 50,000 to 200,000 epidemic curves by randomly selecting values of these four parameters from plausible distributions. Each of these curves is tested to see how well it fits the surveillance and survey data. A sample of curves is drawn from the full set with the likelihood of selection proportional to the goodness of fit. The result is a most likely curve that provides a point estimate of prevalence in each year and a range around the point estimate.The estimates of adult HIV prevalence are used in the AIDS module of Spectrum The rate of progression from infection to AIDS death for adults is a distribution drawn from cohort studies in sub-Saharan Africa People at any stage are subject to non-AIDS mortality at the same rates as those who are not infected. The progression of children from infection to AIDS death is modeled as a double Weibull curve fitted to longitudinal data Spectrum estimates the uncertainty around the estimate of each indicator by using a Monte Carlo approach. One thousand projections are calculated using different prevalence curves provided by EPP and drawing random values of other key parameters  for each projection. The results are analyzed to produce 5% and 95% plausibility bounds around each point estimate. Results for key HIV/AIDS indicators are shown in HIV trends at antenatal surveillance sites are shown for urban and rural sites in When these curves are adjusted to match the national population survey of 2004 and weighted by the adult urban and rural populations they suggest that national prevalence among all adults 15–49 in Botswana peaked at about 26% in 2001 and has since declined slightly to 25.7% (24.8%–26.7%) by 2007. This implies that 280,000 adults 15–49 are currently infected, 47% in urban areas and 53% in rural areas. While the national survey in 2004 found that the peak ages for prevalence were 30–34 for both men and women, high levels of prevalence persist up to ages 70–74. When adult infections over the age of 49 are included the total number of adults infected with HIV is estimated to be about 310,000 .The prevalence trend combined with the pattern of progression from new infection to AIDS death indicates that the annual number of new adult infections peaked at around 33,000 in 1995 and declined to about 20,000 by 2002 and is currently around 18,000 .Incidence among those 15–49 is declining, from 3.5% in 2000 to 2.4% in 2007. While the overall population growth rate in Botswana has declined to about 1.7% today, the annual growth rate among all adults is still 2.4%. Therefore, the overall absolute number of new infections each year is approximately constant.The annual number of adult AIDS deaths rose steadily during the 1980s and 1990s to a peak of nearly 16,000 in 2003. Without any ART program adult AIDS deaths would have continued increasing to nearly 23,000 in 2007. However, Botswana has implemented a vigorous treatment program that has expanded the number of adults receiving ART from 932 in 2000 to 85,497 by the end of 2007, about 83% of need Those currently receiving ART will continue to need it for many years to come. In addition, about 23,000 adults newly progress to need for ART each year. As a result the need for ART is expected to increase by almost 60% from 120,000  in 2007 to about 190,000 by 2016 if the high levels of coverage are maintained. This has important cost and sustainability implications for the programme.The high levels of HIV infection in adults mean that many children are exposed to the risk of acquiring infection from their mothers. The estimated number of new infections among children peaked at around 4600 in 1999. PMTCT services were introduced in 2002 and coverage expanded rapidly. By 2007 91% of HIV-positive pregnant women received antiretrovirals to reduce the risk of mother-to-child transmission HIV-positive children have benefited from an expansion of treatment programs. By the end of 2007 7,400 children were receiving ART, nearly all those estimated to be in need, and almost 10,000 were receiving co-trimoxazole. As a result child AIDS deaths have been reduced from about 3,000 in 2001 to 790 (600–1020) in 2007. Nearly 11,000 child deaths have been averted by the combined effects of PMTCT, child treatment and adult ART.As a result of adult AIDS deaths a significant proportion of children have lost one or both parents. We estimate that there were 130,000  orphans in 2007, about 16% of all children under the age of 18. Three-quarters of these orphans are due to AIDS. One-third of households caring for orphans are receiving external support Botswana has an HIV surveillance system and a national HIV survey that provide the basis for estimates of the extent of the HIV epidemic and its dynamics. These data indicate that prevalence is currently declining slightly in urban areas and stable in rural areas. The estimates produced from these data indicate that the number of new infections rose rapidly during the early 1990s, peaking in the mid-1990s before falling to a stable level of about 18,000 new infections per year today. The number of AIDS deaths started to grow rapidly about 10 years later than the rise in new infections, peaking just before the expansion of ART.The successful expansion of the ART program has increased coverage to over 80% of those in need of treatment. This expansion has had significant benefits, reducing the annual number of AIDS deaths by half and, as a consequence, also cutting in half the number of new orphans each year. The high coverage of adult ART has also contributed to a reduction in mother-to-child transmission of HIV.Due to the high number of new infections in the past 24,000 adults progress to eligibility for ART each year. As a result, the need for adult ART will increase by nearly 60% by 2016. This presents a major challenge to maintain the current levels of high coverage.The PMTCT program represents a major success with over 90% of HIV-positive women receiving antiretrovirals to prevent transmission of HIV to their children. The program has averted an estimated 10,000 child infections since its inception. The combined effects of the PMTCT program and the child treatment program have averted an estimated 11,000 child AIDS deaths. With fewer new child infections the need for child treatment is also be reduced.Unfortunately similar progress has not been made in reducing the number of new adult infections . Some pr


In the present study, we investigated the role of met expression on the modulation of apoptosis in colorectal tumours. The gene expressions of c- met and the anti-apoptotic bcl -2 family, including bcl -2, bcl -x L and bcl-w, were analysed in human colorectal adenomas and adenocarcinomas by using a quantitative polymerase chain-reaction combined with reverse transcription. In seven of 12 adenomas and seven of 11 carcinomas, the c- met gene was overexpressed. The bcl -w, bcl -2 and bcl -x L genes were over-expressed in nine, five and six of 12 adenomas and in five, two and seven of 11 carcinomas, respectively. The c- met mRNA level in human colorectal adenomas and carcinomas was correlated with bcl -w but not with bcl -2 or with bcl -x L mRNA level. The administration of c- met -antisense oligonucleotides decreased Met protein levels in the LoVo human colon cancer cell line. In the case of c- met -antisense-treated cells, apoptotic cell death induced by serum deprivation was more prominent, compared to control or c- met -nonsense-treated cells. Treatment with c- met -antisense oligonucleotides inhibits the gene expression of bcl -w in LoVo cells. On the other hand, the gene expression of bcl -2 or bcl -x L was not affected by treatment with c- met -antisense oligonucleotides. These findings suggest that Met expression modulates apoptosis through bcl -w expression in colorectal tumours. © 2000 Cancer Research CampaignThe


Households experience HIV and AIDS in a complex and changing set of environments. These include health and welfare treatment and support services, HIV-related stigma and discrimination, and individual and household social and economic circumstances. This paper documents the experiences of 12 households directly affected by HIV and AIDS in rural KwaZulu Natal, South Africa, between 2002 and 2004. The households were observed during repeated visits over a period of more than a year by ethnographically trained researchers. Field notes were analysed using thematic content analysis to identify themes and sub-themes. This paper focuses on three dimensions of household experience of HIV and AIDS that have received little attention in HIV and AIDS impact studies. First, that experience of HIV and AIDS is cumulative. In an area where population surveys report HIV prevalence rates of over 20% in adults, many households face multiple episodes of HIV-related illness and AIDS deaths. We describe how these challenges affect perceptions and responses within and outside households. Second, while over 50% of all adult deaths are due to AIDS, households continue to face other causes of illness and death. We show how these other causes compound the impact of AIDS, particularly where the deceased was the main income earner and/or primary carer for young children. Third, HIV-related illness and AIDS deaths of household members are only part of the households’ cumulative experience of HIV and AIDS. Illness and death of non-household members, for example, former partners who are parents of children within the households or relatives who provide financial support, also impact negatively on households. We also discuss how measuring multiple episodes of illness and deaths can be recorded in household surveys in order to improve quantitative assessments of the impact of HIV and AIDS. Families and households in rural South Africa face the adverse demographic, social and economic consequences of the severe HIV epidemic. In areas of rural KwaZulu Natal where the prevalence of HIV in adults (15–54 years) had reached 23% in 2003/2004 , househoIn no society do households exist in isolation. In South Africa the Apartheid-era control of settlement mainly of the African population and the entrenched migrant labour system has resulted in the phenomenon of “stretched” households . In the To explore the impact of HIV and AIDS on rural households in an area with a very rapid and severe HIV epidemic, we conducted a two and a half year ethnographic study of 20 households in rural South Africa. In this paper we describe the past and current experiences of, and responses to, multiple illness and death in 12 of the study households, which were observed for more than one year. Life histories collected from household respondents included information about events occurring in other households that are linked to the households investigated here through kinship or social ties. We also discuss ways of modifying household surveys in order to improve quantitative assessments of the impact of HIV and AIDS.This study was conducted in the northern KwaZulu Natal, South Africa. The area includes land under the Zulu tribal authority, formerly part of a homeland under the Apartheid-era Bantu Authorities Act of 1951 , and an KwaZulu Natal is the province with South Africa's highest HIV prevalence rate among antenatal clinic attendees. A 1998 antenatal survey in the largest clinic within study area found that 41% of pregnant women were HIV infected  . In 2003This paper presents an analysis of field notes collected between 2002 and 2004 as part of a study examining the experiences of households directly affected by HIV and AIDS. The study collected ethnographic data through close observation by research staff 20 households caring for at least one adult with disease symptoms indicative of AIDS. Households were identified by volunteers in a local home-based care programme, by research nurses visiting households in the area, nurses or through opportunistic contacts by research assistants in the course of their fieldwork.Depending on events taking place in the study households, the three research assistants might sometimes spend several days with one particular household or person, while at other times they would visit for an hour or so every couple of weeks for an update. Research assistants were flexible in taking part in household activities: they went with respondents to hospital and welfare services, joined them on visits to neighbours or the market, and attended and helped during funerals. In addition, they accompanied home-based care volunteers on their household visits as well as attended their meetings and training sessions. Some study participants and volunteers actively updated the research assistants themselves, for example, letting them know about hospital admissions. Two of the three research assistants lived in the same community and were in daily contact with many volunteers and household members. All were women and spoke fluent Zulu and English. Two of them had post-graduate-level education; all were trained in ethnographic methods. Moitse, one of this paper's co-authors, had previous ethnographic study experience. Field notes were written up immediately after visits to study households, and were managed and coded using the N6 software (QSR).The analysis presented in this paper is based on detailed field notes from visits to 12 of the 20 households that were observed for more than 1 year. Information on illness and death of non-household members who were connected to the household was also collected. These field notes were grouped into themes and sub-themes as they emerged in order to explore both content  and discourse . Pseudonyms are used throughout this paper.As no clinical tests were conducted during fieldwork, individuals’ HIV status and a diagnosis of AIDS are based on respondents’ self-reports of having received a positive test result , the reports of other household members, volunteers, or on our own interpretation given the signs and symptoms exhibited immediately prior to death. During the period of this study, no antiretroviral treatment programmes were being provided in the area by the government health services or home-based care voluntary organizations.Prior to starting fieldwork the study was discussed with the Community Advisory Board of the Africa Centre for Health and Population Studies; ethical approval was granted by the Faculty of Medicine Ethics Committee of the University of KwaZulu Natal. The head and other senior members of the household gave verbal consent allowing research assistants to visit and subsequently make notes about their observations and conversations. The research team provided support to the study households in a variety of ways including arranging transport in emergencies and introducing households to home-based care volunteers and the Africa Centre social worker.When we first met the households their daily lives were dominated by efforts to cope with the direct and indirect effects of having a sick member with symptoms indicative of AIDS. In seven households, one or more members died of AIDS during the study. For most households, the episode of illness that triggered our initial visits was only a small part of their cumulative experience of HIV and AIDS. Half of the households had experienced the death from AIDS of another member before they were enrolled in the study. Household responses to HIV-related illness and AIDS deaths were conditioned by other historical and contemporary circumstances and events. These predominantly poor households faced insecurity in many aspects of their lives, and adverse consequences of multiple experiences of HIV and AIDS were often compounded by other events, for example, additional episodes of illness or death due to other causes. In addition, the impact of HIV-related illness and AIDS deaths of closely connected people outside the household had social, emotional and economic repercussions for the households.Mrs. Ntombela, a divorced pensioner in her 60s, openly discussed how HIV and AIDS had affected her family. The household circumstances at the time of enrolment and at the end of the study are shown in As this case illustrates, households in this area are frequently extended with three generations living together. A common arrangement is for a household headed by older people to also include their unmarried adult children and their grandchildren. Thus, in a population with a generalized HIV epidemic and very high HIV prevalence, extended households may have to respond to the illness and death of more than one adult sibling, their partners, and their young children. Repeated deaths led to increases in the dependency ratio of households. In all households where an adult member had died before the study started, the deceased had been a parent. Thus, households were already caring for one or more orphaned child at the time another adult died. Many of the caregivers were young and were also HIV positive. Thus, some children lost not only one or both parents but their subsequent primary caregiver too.The economic resources of all households experiencing repeated episodes of illness and death and an increased dependency ratio were severely affected. Increases in expenditure on health care and funerals often came at the same time as losing the income from the patient or their carer and result in deepening financial difficulties. Households in our study often found themselves unable to afford health care, schooling, or adequate food, and several borrowed money. The situation was most acute before and immediately after a death because of the difficulty in obtaining government cash grants for disability and caring for orphans. The delay in obtaining these grants is due in part to the time it takes to complete the application process. As well as because some of the poorest households did not have enough money to apply for a grant, for example to pay for transport to health and welfare offices or in some cases, sadly, to pay the bribes demanded in order to supply documents or process applications. Case studies detailing the experience of the study households in applying for, and receiving grants have been published elsewhere .diseased’ and ‘poor’. Several respondents felt strongly that their impoverished circumstances deterred people from visiting or helping them out, and that poverty exacerbated the stigma around HIV and AIDS. A succession of deaths among previous sexual partners was often seen by family and neighbours as a clear sign that the deceased and surviving partners were HIV positive, and was accompanied by considerable discussion about sexual behaviour. Such intense scrutiny of their partnerships and sexual behaviour was distressing to surviving partners, some of whom felt criticized, blamed, and stigmatised. This is illustrated by the experience of Mr. Nsele described in case study 2.There were also psycho-social consequences for households experiencing multiple HIV-related illness and AIDS deaths that evolved more gradually, in particular stigma and discrimination. Over time, with each new episode of illness and death, respondents felt increasingly isolated and stigmatised by relatives and neighbours. When describing how they believed that they were seen by neighbours and service providers, respondents used stigmatizing labels such as ‘evil things’ he had done to their daughter. Tensions between the families were somewhat eased by his forthright mother, who stated publicly at his funeral that nobody had killed her son rather that he had died from AIDS.Mr. and Mrs. Nsele were living with their four children at the start of the study. Mrs. Nsele was already very sick and was being cared for primarily by her husband until he also became ill. Although it is customary for neighbours to visit households affected by illness, the family felt ostracized, with the only visitors being one neighbour, Mr. Nsele's mother and a home-based care volunteer. After his wife died, Mr. Nsele became increasingly conscious that his past sexual behaviour was being talked about and that he was accused of infecting his wife. His perception seemed to be well-founded: home-based care volunteers and neighbours related stories about his past escapades and extra-marital relationships to the study team. They were highly critical, some even suggesting that his poor health was ‘deserved’. As he sickened, Mr. Nsele felt unsupported and stigmatized because of his wife's death, his poverty and his own ill-health. He responded by shunning most offers of help and relying increasingly on his teenage sons. He was less welcoming of visits by the research team. After his death, his own relatives became suspicious that his wife's family had caused his death through bewitchment because of the ‘While perceived and enacted stigmatizing behaviour towards individuals and households affected by HIV and AIDS was common, pre-existing tensions and conflicts with relatives and neighbours influenced the form that such behaviour took. Many people who disclosed that they, or the person they cared for, were HIV positive, believed that they had been infected because of witchcraft by someone outside the family. Often poor relationships between ex-partners or the family of ex- and deceased partners deteriorated further as families placed the ‘blame’ for HIV infection on one another. Feeling bewitched added to the sense of helplessness and victimization expressed by many households who had experienced multiple deaths in quick succession.While the HIV epidemic has resulted in a dramatic rise in adult mortality over the last two decades in the study area, nearly half of all adult deaths are due to causes other than AIDS . ConsequThe Msweli household was repeatedly hit by illness, death and disability before and during the study. At the start of the study, the household consisted of Rose, a widowed pensioner, her adult daughter Zodwa, and five grandchildren, none of whose parents were members of the household. Three children were orphans. Two of Rose's adult children had died. One daughter had died of AIDS leaving Jacob who was 8 years old. Jacob was also HIV positive and suffered from repeated bouts of tuberculosis and eye infections. Zodwa, his aunt, was Jacob's primary caregiver. Jacob's father lived elsewhere; he was sick and unable to send money for Jacob. Zodwa and Jacob were very attached to each other. Zodwa spent a lot of time and money taking Jacob to see different doctors. She was supported in doing this by her boyfriend and her mother who helped pay for some of the health care and transport costs. During the study Zodwa too was diagnosed as HIV positive and started to become ill.The toll that HIV and AIDS took on this household was compounded by illness and death due to other causes. Two of the grandchildren in this household were 12-year-old twins, abandoned a few years before by their mother and taken in by Rose. One twin had severe mental and physical disabilities and required constant care, the other had moderate learning and behavioural difficulties. Zodwa had been injured 2 years before in a car accident. During the study, Rose had a stroke and was hospitalized for several weeks. The family felt that Rose's stroke had been brought about by the stress of repeated traumas in the family. Shortly afterwards, Phumzile, another married daughter of Rose's moved into the household. Phumzile had previously been living in Johannesburg but had been ill for some time with haemophilia. She moved with her young sons to live with her mother after her husband refused to care for her anymore. She died within a month of arriving due to complications. The role of caring for the numerous sick and dependent children and adults moved back and forth between Rose and Zodwa as they struggled with own health problems.Non-AIDS disability, illness or death can have the same or a greater economic and social impact than AIDS depending on the person affected. Illness and deaths of older people were common in many households and, given their pivotal role in the households they belonged to, their deaths resulted in major changes for households. Five of the households studied were headed by someone over 50 years. All the older women in the study were the primary caregiver of at least one grandchild; in some cases because the children had been orphaned but in others because their parents were absent from the household for employment-related or other reasons. In addition to their role as caregivers, several households were financially dependent on an older person's government pension.Many household respondents and volunteer caregivers described their distress with having to respond to seemingly relentless bouts of severe illness and repeated bereavements. Past experiences primed carers to expect that death was inevitable. Some respondents focused their expressions of fear about the future on worries about financial and practical problems. Others dwelt on the reasons why they were being repeatedly affected by illness and death. Mthokozi, a 5-year-old whose mother had died of AIDS a year before, reflected the general mood in the Msweli household when he talked about his sick aunt: “My mother was sick like Zodwa and I think that Zodwa will die because her sickness is the same as for my mother.” Another adult respondent added: “What have we done wrong in this household? Months back Phumzile died and now Zodwa is sick.”The impact of HIV and AIDS was not restricted to experiences within the household. HIV-related illness and AIDS deaths of people who lived elsewhere, but were connected to the households studied, also had repercussions on study households. When discussing the household's experience of HIV and AIDS respondents often explained a situation by describing events, so to speak, ‘off-stage’.The Gebashe household was large. Headed by Mr. Gebashe, it included his wife, seven children and five grandchildren. The household composition at the start and end of the study are shown in Illnesses or deaths occurring in one household can result in people moving to another household motivated by the need for care or financial assistance, thus changing the composition and dependency ratio of both households. At the start of the study, almost all the households included children whose parents were not married or co-habiting. Although in most cases little or no financial and material support was given by former partners or parents outside the household, such assistance as was given typically ceased when the person became ill or died because the deceased person's relatives were unwilling to continue making maintenance payments. Therefore, this support stopped at the same time that the affected household was undergoing extreme financial difficulties due to income loss, increased costs, and while waiting to receive government grants. This not only exacerbated household poverty but also fostered respondents’ feelings that they were unsupported and stigmatized, and heightened pre-existing conflicts between households.The households in this study experienced repeated episodes of illness and death during two and a half years of observation. These shocks were often closely followed by or occurred at the same time as other experiences of HIV and AIDS within the household or in inter-connected households. The age-specific patterns of sexual and vertical transmission of HIV, together with the social context in rural South Africa, in which households are commonly extended to include unmarried, adult children, create a situation in which multiple HIV-related illness and AIDS deaths are likely to occur. Such repeated episodes pushed these generally poor households into deepening financial crisis. The increased expenditure due to illness and death leading to food insecurity and problems with keeping children in school have also been reported by other studies conducted in Africa . The deaMultiple experiences of HIV-related illness and AIDS deaths, compounded by other causes of death, elicited strong emotional responses from surviving household members. Distress about the apparently relentless way in which their families were being struck by illness and death added to their fear of HIV and AIDS stigma, exacerbating their feelings of isolation and lack of support from relatives and neighbours. The study team observed that neighbours and home-based care volunteers considered multiple deaths of young people to be ‘proof’ that a household had members who were HIV positive. Neighbours who had previously made visits to see young people when they were ill, reported becoming concerned about HIV when other members of the household also fell ill and consciously ceasing to visit.Our observations suggest that while AIDS is an important cause of ill-health and mortality in young adults and children, illness and death due to other causes compound the negative consequences of the HIV epidemic. In some cases, the effect of these other deaths on the household was critical, particularly when the person who died was a pivotal member of the household, such as a senior member of the family or an older pensioner. The qualitative data provide contextual information in addition to findings from an quantitative study of longitudinal, population-based data in the same area that showed that multiple adult deaths, independent of cause, are significantly predictive of household dissolution and migration . The robThe study has also highlighted that households are not isolated from events occurring in their immediate social networks as well as elsewhere in the community. Adult deaths in one household may have repercussions for several other households connected to this household. The results of this qualitative study have implications for the design and interpretation of studies designed to quantify the impact of AIDS deaths on households. As suggested by off-stage presents substantial challenges for systematic recording and introduces complexities in data management and analysis. Thus, a recent paper has explicitly called for greater use of case studies as an approach that captures household experiences and responses more holistically (Our finding that a household's experience of HIV and AIDS is only partially revealed without information about events outside the household is probably the most difficult to address in population-based surveys and other questionnaire-based studies . Such instically . However


A record-linkage study was carried out between the Italian AIDS Registry and 24 Italian cancer registries to compare cancer excess among persons with HIV/AIDS (PWHA) before and after the introduction of highly active antiretroviral therapy (HAART) in 1996. Standardised incidence ratios (SIR) were computed in 21951 AIDS cases aged 16–69 years reported between 1986 and 2005. Of 101 669 person-years available, 45 026 were after 1996. SIR for Kaposi sarcoma (KS) and non-Hodgkin lymphoma greatly decreased in 1997–2004 compared with 1986–1996, but high SIRs for KS persisted in the increasingly large fraction of PWHA who had an interval of <1 year between first HIV-positive test and AIDS diagnosis. A significant excess of liver cancer (SIR=6.4) emerged in 1997–2004, whereas the SIRs for cancer of the cervix (41.5), anus (44.0), lung (4.1), brain (3.2), skin , Hodgkin lymphoma (20.7), myeloma (3.9), and non-AIDS-defining cancers (2.2) were similarly elevated in the two periods. The excess of some potentially preventable cancers in PWHA suggests that HAART use must be accompanied by cancer-prevention strategies, notably antismoking and cervical cancer screening programmes. Improvements in the timely identification of HIV-positive individuals are also a priority in Italy to avoid the adverse consequences of delayed HAART use. Three types of cancer that occur in HIV-positive individuals, namely Kaposi sarcoma (KS), non-Hodgkin lymphoma (NHL), and invasive cervical cancer (ICC), are currently included in the European clinical AIDS definition . HoweverAfter the introduction of the highly active antiretroviral therapy (HAART) in 1996, huge declines in KS and NHL incidence have been consistently reported in high-resource countries  were diagnosed with AIDS between 1986 and 2005; (2) were aged between 16 and 69 years at the time of AIDS diagnosis and (3) reported a ‘legal residence’ in areas covered by a CR. Person-years at risk were computed between 5 years prior to AIDS diagnosis, and the date of cancer or death or 10 years after AIDS diagnosis, whichever occurred earlier. This interval was left or right censored if no complete CR data were available for the corresponding years. To reduce losses to follow-up, dates of death were updated through record linkage with the National Mortality Database.Observed cases included incident cancer cases reported to CRs during the above-defined person-years at risk. Cancer site and type were classified according to the International Classification of Disease, 10th revision  and wereExpected numbers of different cancers were computed for each CR from sex-, age-, and period-specific incidence rates .SIR were calculated for calendar period, distinguishing the pre-HAART (1986–1996) from the post-HAART (1997–2004) period. For 1997–2004, and for cancers showing a significantly increased risk and at least 10 cases, SIRs were also computed separately by age group , HIV transmission category (injecting drug users (IDUs), men who have sex with men (MSM), heterosexuals) and time of cancer occurrence in respect to AIDS diagnosis . SIR were also computed separately by interval between first HIV-positive test and AIDS diagnosis , and country of birth .A total of 21 951 AIDS cases (78% men and 22% women) were reported in Italy between 1986 and 2005 in areas covered by a CR . The numA significantly elevated risk emerged in 1997–2004 for cancer of the liver  and penis , whereas the excess risk for leukaemia disappeared. Elevated SIRs for cancer of the anus , vulva and vagina , lung , brain , skin , HL , and multiple myeloma  in 1997–2004 were similar to those found in 1986–1996 . The comPersons with HIV/AIDS born outside Italy contributed 15% of person-years and 7.8% of cancer cases in 1997–2004. They showed similar SIR for AIDS-defining illnesses and slightly lower SIR of non-AIDS-defining cancers  than PWHA born in Italy (data not shown).Microscopic confirmation was available after 1996 for all ICC, anal cancer, and HL , as well as 79% of lung cancer. Eleven out of 16 liver cancers were microscopically or instrumentally confirmed. Microscopic confirmation was available for only one (a glioma) out of eight brain tumours, and seven had a concomitant AIDS-defining illness in the brain .For both AIDS- and non-AIDS-defining cancers the highest SIR emerged in the 3 months prior to or after AIDS diagnosis . Prior tSIR years for KS, NHL, and HL were lower among PWHA younger than 35 compared with older ones, whereas those for non-AIDS-defining cancers other than HL were higher . Women sPersons with HIV/AIDS who had less than 1-year interval between first HIV-positive test and AIDS diagnosis differed from other PWHA in many ways . Among tOur study showed substantial changes in the cancer pattern of Italian PWHA after the introduction of HAART in 1996. Non-Hodgkin lymphoma replaced KS as the most frequent cancer type and non-AIDS-defining cancers increased from 15 to 25% of all cancers. For the first time a significant excess of liver cancer emerged in the Italian AIDS linkage study  continued to be significantly increased among PWHA after 1996. The greatest cancer excess was found in proximity to AIDS diagnosis, but persisted in the 10 years afterwards. Notably, elevated SIRs were seen overall and in each HIV transmission category for cancers of the cervix and the anogenital tract that are, in the vast majority, associated with HPV infection . Hence, With respect to HL, our findings confirm previous reports , but an An increased risk for lung cancer among Italian PWHA was also confirmed (Skin cancer (non-melanoma) was increased by two-fold in PWHA as in previous reports  of information on the date of first HIV-positive test in the AIDS Registry allowed us, however, to focus on PWHA who had concomitant, or nearly concomitant, HIV infection and AIDS-defining illness. Such late presenters increased in Italy from 20.5% in 1996 to 55.5% of AIDS cases in 2007 . They weOur study showed that to prevent cancer in PWHA with increasing life expectancy, the use of HAART must be accompanied by more effective cancer-prevention strategies (