Evaluation of the feasibility and acceptability of the 'Care for Stroke' intervention in India, a smartphone-enabled, carer-supported, educational intervention for management of disability following stroke.

OBJECTIVES: (1) To identify operational issues encountered by study participants in using the 'Care for Stroke' intervention; (2) to evaluate the feasibility and acceptability of the intervention. DESIGN: Mixed-methods research design. SETTING: Participant's home. Participants were selected from a tertiary hospital in Chennai, South India. PARTICIPANTS: Sixty stroke survivors treated and discharged from the hospital, and their caregivers. INTERVENTION: 'Care for Stroke' is a smartphone-enabled, educational intervention for management of physical disabilities following stroke. It is delivered through a web-based, smartphone-enabled application. It includes inputs from stroke rehabilitation experts in a digitised format. METHODS: Evaluation of the intervention was completed in two phases. In the first phase, the preliminary intervention was field-tested with 30 stroke survivors for 2 weeks. In the second phase, the finalised intervention was provided to a further 30 stroke survivors to be used in their homes with support from their carers for 4 weeks. PRIMARY OUTCOMES: (1) operational difficulties in using the intervention; (2) feasibility and acceptability of the intervention in an Indian setting. Disability and dependency were assessed as secondary outcomes. RESULTS: Field-testing identified operational difficulties related to connectivity, video-streaming, picture clarity, quality of videos, and functionality of the application. The intervention was reviewed, revised and finalised before pilot-testing. Findings from the pilot-testing showed that the 'Care for Stroke' intervention was feasible and acceptable. Over 90% (n=27) of the study participants felt that the intervention was relevant, comprehensible and useful. Over 96% (n=29) of the stroke survivors and all the caregivers (100%, n=30) rated the intervention as excellent and very useful. These findings were supported by qualitative interviews. CONCLUSIONS: Evaluation indicated that the 'Care for Stroke' intervention was feasible and acceptable in an Indian context. An assessment of effectiveness is now warranted.

The protein tyrosine phosphatase, Shp2, positively contributes to FLT3-ITD-induced hematopoietic progenitor hyperproliferation and malignant disease in vivo.

Internal tandem duplications (ITDs) in the fms-like tyrosine kinase receptor (FLT3-ITDs) confer a poor prognosis in acute myeloid leukemia (AML). We hypothesized that increased recruitment of the protein tyrosine phosphatase, Shp2, to FLT3-ITDs contributes to FLT3 ligand (FL)-independent hyperproliferation and STAT5 activation. Co-immunoprecipitation demonstrated constitutive association of Shp2 with the FLT3-ITD, N51-FLT3, as well as with STAT5. Knockdown of Shp2 in Baf3/N51-FLT3 cells significantly reduced proliferation while having little effect on WT-FLT3-expressing cells. Consistently, mutation of N51-FLT3 tyrosine 599 to phenylalanine or genetic disruption of Shp2 in N51-FLT3-expressing bone marrow low-density mononuclear cells reduced proliferation and STAT5 activation. In transplants, genetic disruption of Shp2 in vivo yielded increased latency to and reduced severity of FLT3-ITD-induced malignancy. Mechanistically, Shp2 co-localizes with nuclear phospho-STAT5, is present at functional interferon-γ activation sites (GAS) within the BCL2L1 promoter, and positively activates the human BCL2L1 promoter, suggesting that Shp2 works with STAT5 to promote pro-leukemogenic gene expression. Further, using a small molecule Shp2 inhibitor, the proliferation of N51-FLT3-expressing bone marrow progenitors and primary AML samples was reduced in a dose-dependent manner. These findings demonstrate that Shp2 positively contributes to FLT3-ITD-induced leukemia and suggest that Shp2 inhibition may provide a novel therapeutic approach to AML.

Antiapoptotic function of NF-kappaB in T lymphocytes is influenced by their differentiation status: roles of Fas, c-FLIP, and Bcl-xL.

Inducible protection from apoptosis in vivo controls the size of cell populations. An important question in this respect is how differentiation affects mechanisms of apoptosis regulation. Among mature T lymphocytes, the NF-kappaB/Rel transcription factors are coupled to receptors that control cell population sizes by concurrently regulating survival and multiplication. In the present study, we used a transgenic inhibitor of NF-kappaB/Rel signaling to investigate the role of this pathway in proliferation and death of mature T cells in vivo. The results indicate that NF-kappaB integrates two critical yet distinct molecular pathways preventing apoptosis affected by the death receptor Fas, coordinately regulating levels of FLIP and Bcl-x(L) in primary T cells. Surprisingly, NF-kappaB blockade preferentially impacted naive as compared to memory T cells. The Fas/FasL pathway was linked to these findings by evidence that the abnormalities imposed by NF-kappaB inhibition were ameliorated by Fas deficiency, particularly for the CD4(+) lineage. Moreover, levels of an inhibitor of Fas-mediated apoptosis, c-FLIP, were diminished in cells expressing the transgenic inhibitor. NF-kappaB was also linked to T cell survival in vivo by mediating induction of Bcl-x(L): restoration of Bcl-x(L) levels reversed the preferential deficit of naive T cells, differentially impacting the CD4 and CD8 subsets. These results show that promoting survival and effective multiplication are central roles for NF-kappaB in T lymphoid homeostasis in vivo, but this effect and its underlying mechanisms are influenced by the developmental state of the lymphocyte.

Unfolding and refolding of a quinone oxidoreductase: alpha-crystallin, a molecular chaperone, assists its reactivation.

alpha-Crystallin, a member of the small heat-shock protein family and present in vertebrate eye lens, is known to prevent the aggregation of other proteins under conditions of stress. However, its role in the reactivation of enzymes from their non-native inactive states has not been clearly demonstrated. We have studied the effect of alpha-crystallin on the refolding of zeta-crystallin, a quinone oxidoreductase, from its different urea-denatured states. Co-refolding zeta-crystallin from its denatured state in 2.5 M urea with either calf eye lens alpha-crystallin or recombinant human alpha B-crystallin could significantly enhance its reactivation yield. alpha B-crystallin was found to be more efficient than alpha A-crystallin in chaperoning the refolding of zeta-crystallin. In order to understand the nature of the denatured state(s) of zeta-crystallin that can interact with alpha-crystallin, we have investigated the unfolding pathway of zeta-crystallin. We find that it unfolds through three distinct intermediates: an altered tetramer, a partially unfolded dimer, which is competent to fold back to its active state, and a partially unfolded monomer. The partially unfolded monomer is inactive, exhibits highly exposed hydrophobic surfaces and has significant secondary structural elements with little or no tertiary structure. This intermediate does not refold into the active state without assistance. alpha-Crystallin provides the required assistance and improves the reactivation yield several-fold.

IL-4 signaling, gene transcription regulation, and the control of effector T cells.

The central goal of our laboratory is to understand the regulation of lymphoid cells through molecular mechanisms of signal transduction and transcriptional control. A long-standing focus has been on changes that influence the effector function of mature lymphocytes. Work in the laboratory is oriented toward the identification of new regulatory mechanisms using cell lines and primary cells, and the validation of these in vitro findings in mouse models of immune responses and diseases. In this review, we summarize key insights into the regulation of T helper cell function during the phase of immunity where effector responses arise de novo. Particular interest has been centered on cytokine gene regulation as part of T cell differentiation into the Th1 and Th2 subsets. Information on IL-4 receptor signaling and the role of NF-kappaB transcription factors is reviewed. Our more recent work is designed to understand how regulation at the Th1/2 effector stages is related to the control of memory T cell survival, immune recall responses, and the role of these responses in immune-mediated disease.

Expression of recombinant zeta-crystallin in Escherichia coli with the help of GroEL/ES and its purification.

zeta-Crystallin is a taxon-specific crystallin found in the eye lens of guinea pig and other hystricomorph rodents and camelids. It is an NADPH:quinone oxidoreductase and is also present in low amounts in other tissues where it might act as a detoxifying enzyme. A lens-specific promoter confers lens-specific expression of the gene in high amounts where it is speculated to play a structural role in maintaining the transparency of the lens ensemble. A deletion mutation leads to autosomal dominant congenital cataract and also results in the loss of NADPH binding. In order to perform structural studies with the protein with an aim to delineate the cause of cataract in these mutant guinea pigs, recombinant zeta-crystallin was cloned and expressed in Escherichia coli. The overexpression of the protein in E. coli resulted in a major fraction of it partitioning into inclusion bodies. The co-overexpression of the bacterial chaperone system GroEL/ES along with zeta-crystallin could significantly enhance the yield of soluble protein. Active zeta-crystallin could then be purified from the E. coli using Mono Q anion exchange FPLC and was found to be identical to the native zeta-crystallin isolated from the guinea pig lens with respect to size, spectral properties, and activity.

Inability of chaperones to fold mutant zeta crystallin, an aggregation-prone eye lens protein.

PURPOSE: zeta-crystallin is a quinone oxido-reductase, recruited in the eye lens of hystricomorphic rodents and camels. A deletion mutation constituting the NADPH-binding domain causes congenital cataract in a strain of guinea pigs. The presence of large quantities of a-crystallin, a molecular chaperone, does not provide any protection against this. In order to investigate whether the underlying reason for the lack of protection is the formation of a folding-incompetent protein, we have expressed the mutant protein in a heterologous system along with other known chaperones. METHODS: We expressed the mutant zeta-crystallin in E. coli along with other chaperones such as GroEL/ES and DnaK/DnaJ/GrpE and then analyzed whether these chaperones could increase the amount of protein partitioning into the soluble fraction of E. coli cells. RESULTS: These chaperones were unable to rescue the mutant protein from partitioning into inclusion bodies, although they could increase the yield of soluble wild-type zeta-crystallin. CONCLUSIONS: The deletion of 34 amino acids, constituting the NADPH-binding domain of zeta-crystallin, makes the protein incompetent to fold correctly and thus form insoluble aggregates. It perhaps suggests why the mutant strain of guinea pigs have cataract at birth even though their lenses contain high amounts of alpha-crystallin. This study also shows that certain mutations can render proteins incompetent to fold into soluble molecules despite abundant assistance.

Development of thyroid follicular adenoma on simvastatin therapy.

HMG-CoA reductase inhibitors (lovastatin, simvastatin, fluvastatin, pravastatin) constitute a potent class of cholesterol-lowering agents, which are increasingly being used these days for primary and secondary prevention of atherosclerotic heart disease. Despite having good overall safety and efficacy profiles, these medications can still cause significant adverse effects including transient elevation of hepatic transaminases, myopathy, and rhabdomyolysis. Preclinical studies have demonstrated a potential of neoplasia in rats. However in clinical trials HMG-CoA reductase inhibitors have not been found to be neoplastic in humans. The dosage used in humans is also significantly lower and therefore it is expected to have a good safety margin. But this may not be entirely true considering the mechanism of neoplastic transformation, which is thought to be different in humans as compared to other species. We report a patient, who developed follicular adenoma with prominent Hurthle cell changes after being on simvastatin for three months but not during one year of pravastatin therapy. In elderly female patients with hyperlipidemia requiring pharmacologic treatment, especially those with a prior history of multinodular goiter, one should consider using an agent which has not been shown to cause thyroid tumors even in animal models. Patients should continue to be followed with frequent periodic thyroid palpation in addition to the usual biochemical monitoring required while on these agents.

IL-4-dependent induction of BCL-2 and BCL-X(L)IN activated T lymphocytes through a STAT6- and pi 3-kinase-independent pathway.

Both B and T lymphocytes require ongoing signals to maintain their viability. The pleiotropic cytokine interleukin (IL-) 4 plays an important role in the maintenance of activated T cells, perhaps reflecting induction of the anti-apoptotic genes Bcl-2 and Bcl-X(L). However, it is not known which of the signalling pathways known to link the IL-4 receptor with transcription regulation are required, or if the levels of Bcl-2/X induction under such physiologic conditions are sufficient to account for the anti-apoptotic effects of IL-4. We report here that although blockade of pathways (PI 3-kinase and pp70 S6 kinase) recruited by the IRS-1/2 adaptor proteins inhibited the anti-apoptotic function of IL-4, Bcl-2/X induction were normal. These findings were recapitulated in primary and culture-adapted T cells whose Stat6 signalling pathway also was defective. These results demonstrate that both the Stat6 and PI 3-kinase pathways can be dispensable for Bcl-2/X induction by IL-4, thus suggesting the involvement of an additional signal transduction pathway. Moreover, the preservation of Bcl-2/X induction despite inhibition of the anti-apoptotic function of IL-4 indicates that this cytokine activates additional protective mechanisms.

Natural history of isolated ventricular septal defect in the first five years of life.

BACKGROUND: Isolated ventricular septal defect (VSD) is the most common congenital heart defect. Incidence, prevalence, and clinical outcomes of VSD have been reported to vary significantly in different geographic areas. Spontaneous closure of VSD, in children, by various methods has been described. HYPOTHESIS: This prospective study was undertaken to evaluate natural history of patients with VSD in the first five years of life in the Northeast Tennessee and Southwest Virginia region. METHODS: Between December 1, 1998 and October 31, 1990, 124 infants were diagnosed clinically with isolated VSD. VSDs were classified as muscular, perimembranous, malalignment, or subpulmonic types by 2-dimensional echocardiogram with color flow mapping. Cardiac catheterization and angiocardiography were performed in 14 patients when clinically indicated. These patients were followed for at least five years. RESULTS: Overall spontaneous closure of VSD was 34% at one year and 67% at five years. Twenty-five percent of perimembranous and 4% of muscular VSDs required surgery by five years. The spontaneous closure rate of muscular VSD was twice that of the perimembranous type, though the relative distribution of both types was almost equal. Overall, 22% of children with VSD need follow-up after the fifth year of life. CONCLUSION: The overall clinical outcome of muscular VSD was consistently better than that of the perimembranous type, though 17% of muscular VSDs, irrespective of size, were open at 5 years of age and needed long-term follow-up.

An unusual peripheral vascular response to dopamine in a neonate.

We report a case of a neonate who developed hypotension immediately after birth, and needed dopamine infusion to sustain his blood pressure and tissue perfusion. He developed cyanosis of his extremity immediately after dopamine was started via peripheral line and improved spontaneously after dopamine was stopped. This happened repeatedly at various sites and at lower concentrations of dopamine. Subsequently, dopamine was replaced by dobutamine and the patient did well. We conclude that some neonates can show heightened alpha-adrenergic response to dopamine and this can lead to ischemic vascular events. Dopamine infusion in neonates should be started at a low-dose via central line.

Paired Stat6 C-terminal transcription activation domains required both for inhibition of an IFN-responsive promoter and trans-activation.

The cytokines IL-4 and IFN-gamma exert biologically antagonistic effects that in part reflect opposing influences on gene transcription. While the molecular mechanisms for IL-4-mediated transcription activation have been extensively studied, little is known about molecular mechanisms required for IL-4 inhibition of IFN-gamma signaling. We have investigated IL-4 inhibition of the IFN-gamma-inducible promoter for IFN regulatory factor-1 (IRF-1). In a cell line with low endogenous Stat6, increasing levels of activated Stat6 at constant doses of IFN-gamma and IL-4 leads to inhibition of the IRF-1 promoter. The Stat1-dependent IFN-gamma activation sequence element of the IRF-1 promoter is a target for Stat6-mediated inhibition despite apparently normal Stat1 DNA binding. However, our data are inconsistent with competition between Stat1 and Stat6 for access to the IRF-1 IFN-gamma activation sequence or for an essential coactivator as a mechanism for this Stat6-mediated inhibition. Instead, the data demonstrate that a threshold of Stat6 transcription activation domains is required for IL-4-dependent inhibition. The findings provide evidence of a novel mechanism in which the Stat6 transcription activation domains play a critical role in the IL-4-mediated inhibition of an IFN-gamma-inducible promoter.

Petit mal seizure in a child with Marfan's syndrome.

We report a case of a 17-year-old white girl with Marfan's syndrome and generalized absence-type seizures since 11 years of age. A video EEG recording for six hours demonstrated 52 episodes of clinical generalized absence-type seizures and three-per-second spike and wave epileptiform discharges, characteristic of petit mal epilepsy. Sodium valproate therapy was successful in controlling her seizures. In this article, we review various possible causes of epilepsy in patients with Marfan's syndrome.

In vivo function of an interleukin 2 receptor beta chain (IL-2Rbeta)/IL-4Ralpha cytokine receptor chimera potentiates allergic airway disease.

Strength of T cell receptor (TCR) signaling, coreceptors, costimulation, antigen-presenting cell type, and cytokines all play crucial roles in determining the efficiency with which type 2 T lymphocytes (Th2, Tc2) develop from uncommitted precursors. To investigate in vivo regulatory mechanisms that control the population of type 2 T cells and disease susceptibility, we have created lines of transgenic mice in which expression of a chimeric cytokine receptor (the mouse interleukin 2 receptor beta chain [IL-2Rbeta] extracellular domain fused to the cytoplasmic tail of IL-4Ralpha) is targeted to the T lymphoid lineage using the proximal lck promoter. This chimera transduced IL-4-specific signals in response to IL-2 binding and dramatically enhanced type 2 responses (IL-4, IL-5, and immunoglobulin E production) upon in vitro TCR stimulation or in vivo antigen challenge. Thus, type 2 effector function was augmented by IL-4 signals transduced through a chimeric receptor expressed in a T cell-specific manner. This influence was sufficient for establishment of antigen-induced allergic airway hyperresponsiveness on a disease-resistant background (C57BL/6).

Protein A induces NO production: involvement of tyrosine kinase, phospholipase C, and protein kinase C.

Protein A of S. aureus exhibits a wide array of immunopotentiating activities. Since the role of nitric oxide (NO) in bioregulation has been well envisaged; we studied the effect of Protein A on NO production by immunocytes both in vivo and in vitro. Our data indicate that PA at a comparable dose of LPS (lipopolysaccharide) increases the NO levels in the serum of Swiss albino mice by about 12-fold from its basal level. The peak level is reached at about 12 hours after i.p. inoculation of PA. However, NO concentration returns to the basal value 15 hours posttreatment. Splenic lymphocytes and peritoneal macrophages showed appreciable increase in NO production when cultured with PA in vitro. Interestingly, inhibitors of tyrosine kinase, phospholipase C, and protein kinase C (PKC) inhibited NO production in splenic lymphocytes. Thus, it appears that these enzymes participate in the signaling cascade induced by PA, which culminates in the production of NO downstream of PKC. It is possible that PA-induced NO production may have relevance with the anti-tumor and anti-parasitic properties of PA, described earlier.