Clinical outcomes of bronchiectasis in India: data from the EMBARC/Respiratory Research Network of India registry.

BACKGROUND: Identifying risk factors for poor outcomes can help with risk stratification and targeting of treatment. Risk factors for mortality and exacerbations have been identified in bronchiectasis but have been almost exclusively studied in European and North American populations. This study investigated the risk factors for poor outcome in a large population of bronchiectasis patients enrolled in India. METHODS: The European Multicentre Bronchiectasis Audit and Research Collaboration (EMBARC) and Respiratory Research Network of India (EMBARC-India) registry is a prospective observational study of adults with computed tomography-confirmed bronchiectasis enrolled at 31 sites across India. Baseline characteristics of patients were used to investigate associations with key clinical outcomes: mortality, severe exacerbations requiring hospital admission, overall exacerbation frequency and decline in forced expiratory volume in 1 s. RESULTS: 1018 patients with at least 12-month follow-up data were enrolled in the follow-up study. Frequent exacerbations (≥3 per year) at baseline were associated with an increased risk of mortality (hazard ratio (HR) 3.23, 95% CI 1.39-7.50), severe exacerbations (HR 2.71, 95% CI 1.92-3.83), future exacerbations (incidence rate ratio (IRR) 3.08, 95% CI 2.36-4.01) and lung function decline. Coexisting COPD, dyspnoea and current cigarette smoking were similarly associated with a worse outcome across all end-points studied. Additional predictors of mortality and severe exacerbations were increasing age and cardiovascular comorbidity. Infection with Gram-negative pathogens (predominantly Klebsiella pneumoniae) was independently associated with increased mortality (HR 3.13, 95% CI 1.62-6.06), while Pseudomonas aeruginosa infection was associated with severe exacerbations (HR 1.41, 95% CI 1.01-1.97) and overall exacerbation rate (IRR 1.47, 95% CI 1.13-1.91). CONCLUSIONS: This study identifies risk factors for morbidity and mortality among bronchiectasis patients in India. Identification of these risk factors may support treatment approaches optimised to an Asian setting.

Stainless steel vs. titanium miniscrew implants: Evaluation of stability during retraction of maxillary and mandibular anterior teeth.

OBJECTIVES: This study was aimed to compare the stability of stainless steel and titanium miniscrew implants of the same diameter and length during en masse retraction of maxillary and mandibular anterior teeth. MATERIALS AND METHODS: Forty miniscrew implants (1.3 mm diameter and 8 mm length) were placed in 10 patients (20 titanium and 20 stainless steel). Stability was checked at insertion (T0), at one month (T1), and at sixth months (T2) and the amount of retraction was recorded in millimeters. RESULTS: Titanium and stainless steel implants were equally stable at the time of insertion. At T1, three titanium miniscrew implants showed grade 2 mobility, whereas seven stainless steel miniscrew implants showed grade 2 mobility. For T2, none of the titanium miniscrew implants had grade 2 mobility while four stainless steel miniscrew implants resulted in grade 2 mobility. Both had an equal frequency of grade 3 and grade 4 mobility. However, the difference in the stability was not statistically significant. No statistical significance was found when the amount of retraction achieved by titanium and stainless steel miniscrew implants was compared between the maxillary and mandibular arches. CONCLUSION: Both titanium and stainless steel miniscrew implants provide good anchorage and remain stable during en masse retraction of maxillary and mandibular anterior teeth. Thus, both miniscrews are clinically effective.

Gene Therapy as the New Frontier for Sickle Cell Disease.

Sickle Cell Disease (SCD) is one of the most common monogenic disorders caused by a point mutation in the ß-globin gene. This mutation results in polymerization of hemoglobin (Hb) under reduced oxygenation conditions, causing rigid sickle-shaped RBCs and hemolytic anemia. This clearly defined fundamental molecular mechanism makes SCD a prototypical target for precision therapy. Both the mutant ß-globin protein and its downstream pathophysiology are pharmacological targets of intensive research. SCD also is a disease well-suited for biological interventions like gene therapy. Recent advances in hematopoietic stem cell (HSC) transplantation and gene therapy platforms, like Lentiviral vectors and gene editing strategies, expand the potentially curative options for patients with SCD. This review discusses the recent advances in precision therapy for SCD and the preclinical and clinical advances in autologous HSC gene therapy for SCD.

Comparison of autologous blood prolotherapy and 25% dextrose prolotherapy for the treatment of chronic recurrent temporomandibular joint dislocation on the basis of clinical parameters: A retrospective study.

Introduction: Prolotherapy is a nonsurgical regenerative injection technique and effective treatment method for the treatment of temporomandibular joint (TMJ) dislocation. Autologous blood and dextrose are commonly used agents for prolotherapy and the aim of this study is to compare the autologous blood injection prolotherapy and 25% dextrose prolotherapy for the treatment of chronic recurrent TMJ dislocation. Method: This is a retrospective cohort study of 20 patients with chronic recurrent TMJ dislocation who were treated by either autologous blood (Group A) or 25% dextrose Prolotherapy (Group B). After prolotherapy, the patients were kept on follow-up and evaluated for maximum mouth opening (MMO), pain at visual analog scale (VAS), mandibular movements, frequency of dislocation, and TMJ sound. The collected data were then statistically analyzed. Results: Group A showed better results in terms of reduction in MMO, mandibular movements as compared to Group B, and a statistically significant difference was found starting from 2 weeks post prolotherapy till 6 months follow-up. Whereas group B showed better results regarding reduction in pain intensity on VAS Scale at all follow-up visits. No statistically significant difference was found between both groups regarding reduction in the frequency of dislocation and TMJ sounds. Conclusion: Both autologous and dextrose prolotherapy gives promising results for the treatment of recurrent TMJ dislocation, however, regarding reduction in MMO and improvement in lateral and protrusive mandibular movements, autologous blood gave better results whereas 25% Dextrose was found to be more effective in terms of reduction of pain in recurrent TMJ dislocation cases.

Recovery from refractory chronic fatigue syndrome with CBT and modafinil.

Many patients with chronic fatigue syndrome (CFS) fail to derive benefit from evidence-based treatments such as cognitive-behavioural therapy (CBT) and graded exercise therapy leading to permanent disability. To discover whether a repeat prescription of modafinil might potentiate the benefits of CBT leading to social recovery as defined by 2 or more point improvement in energy and muscular pain/concentration and return to work or full-time training. Three patients with treatment-resistant CFS (mean duration 17.66 years) treated with modafinil and CBT in a Liaison Psychiatry clinic were retrospectively reviewed. Progress was reviewed at baseline, 4-6 months and 10-24 months. Patients rated their fatigue, pain and concentration using 10-point Likert scales. 2/3 achieved clinically meaningful improvements in energy and pain/concentration and 3/3 achieved social recovery. Modafinil, when prescribed over the medium term, would appear to be a potentially useful potentiating agent when added to CBT.

Combination gene therapy for HIV using a conditional suicidal gene with CCR5 knockout.

BACKGROUND: Gene therapy approaches using hematopoietic stem cells to generate an HIV resistant immune system have been shown to be successful. The deletion of HIV co-receptor CCR5 remains a viable strategy although co-receptor switching to CXCR4 remains a major pitfall. To overcome this, we designed a dual gene therapy strategy that incorporates a conditional suicide gene and CCR5 knockout (KO) to overcome the limitations of CCR5 KO alone. METHODS: A two-vector system was designed that included an integrating lentiviral vector that expresses a HIV Tat dependent Thymidine Kinase mutant SR39 (TK-SR39) and GFP reporter gene. The second non-integrating lentiviral (NIL) vector expresses a CCR5gRNA-CRISPR/Cas9 cassette and HIV Tat protein. RESULTS: Transduction of cells sequentially with the integrating followed by the NIL vector allows for insertion of the conditional suicide gene, KO of CCR5 and transient expression of GFP to enrich the modified cells. We used this strategy to modify TZM cells and generate a cell line that was resistant to CCR5 tropic viruses while permitting infection of CXCR4 tropic viruses which could be controlled via treatment with Ganciclovir. CONCLUSIONS: Our study demonstrates proof of principle that a combination gene therapy for HIV is a viable strategy and can overcome the limitation of editing CCR5 gene alone.

Differential Pathogenicity of SHIV KB9 and 89.6 Env Correlates with Bystander Apoptosis Induction in CD4+ T cells.

SHIV variants KB9 and 89.6 show differential pathogenesis in primate models with KB9 causing rapid CD4 decline while 89.6 failing to induce disease. We attempted to determine whether the differential pathogenicity of KB9 versus 89.6 was a result of differential bystander apoptosis inducing potential (AIP) of the Env glycoproteins from these viruses. We find that the KB9 Env was highly potent at inducing bystander apoptosis in CD4+ target cells compared to 89.6 Env. Cell death induction by KB9 showed classical signs of apoptosis including mitochondrial depolarization, caspase activation and PARP cleavage. Inhibiting Env mediated fusion by T20 peptide inhibited KB9 mediated bystander apoptosis. KB9 and 89.6 differed in terms of co-receptor usage with 89.6 preferring CXCR4 while KB9 using both CXCR4 and CCR5 with equal efficiency. Our study suggests that higher bystander AIP of KB9 Env compared to 89.6 may be the basis for the differential pathogenesis of these viruses.

Bronchiectasis in India: results from the European Multicentre Bronchiectasis Audit and Research Collaboration (EMBARC) and Respiratory Research Network of India Registry.

BACKGROUND: Bronchiectasis is a common but neglected chronic lung disease. Most epidemiological data are limited to cohorts from Europe and the USA, with few data from low-income and middle-income countries. We therefore aimed to describe the characteristics, severity of disease, microbiology, and treatment of patients with bronchiectasis in India. METHODS: The Indian bronchiectasis registry is a multicentre, prospective, observational cohort study. Adult patients (≥18 years) with CT-confirmed bronchiectasis were enrolled from 31 centres across India. Patients with bronchiectasis due to cystic fibrosis or traction bronchiectasis associated with another respiratory disorder were excluded. Data were collected at baseline (recruitment) with follow-up visits taking place once per year. Comprehensive clinical data were collected through the European Multicentre Bronchiectasis Audit and Research Collaboration registry platform. Underlying aetiology of bronchiectasis, as well as treatment and risk factors for bronchiectasis were analysed in the Indian bronchiectasis registry. Comparisons of demographics were made with published European and US registries, and quality of care was benchmarked against the 2017 European Respiratory Society guidelines. FINDINGS: From June 1, 2015, to Sept 1, 2017, 2195 patients were enrolled. Marked differences were observed between India, Europe, and the USA. Patients in India were younger (median age 56 years [IQR 41-66] vs the European and US registries; p<0·0001]) and more likely to be men (1249 [56·9%] of 2195). Previous tuberculosis (780 [35·5%] of 2195) was the most frequent underlying cause of bronchiectasis and Pseudomonas aeruginosa was the most common organism in sputum culture (301 [13·7%]) in India. Risk factors for exacerbations included being of the male sex (adjusted incidence rate ratio 1·17, 95% CI 1·03-1·32; p=0·015), P aeruginosa infection (1·29, 1·10-1·50; p=0·001), a history of pulmonary tuberculosis (1·20, 1·07-1·34; p=0·002), modified Medical Research Council Dyspnoea score (1·32, 1·25-1·39; p<0·0001), daily sputum production (1·16, 1·03-1·30; p=0·013), and radiological severity of disease (1·03, 1·01-1·04; p<0·0001). Low adherence to guideline-recommended care was observed; only 388 patients were tested for allergic bronchopulmonary aspergillosis and 82 patients had been tested for immunoglobulins. INTERPRETATION: Patients with bronchiectasis in India have more severe disease and have distinct characteristics from those reported in other countries. This study provides a benchmark to improve quality of care for patients with bronchiectasis in India. FUNDING: EU/European Federation of Pharmaceutical Industries and Associations Innovative Medicines Initiative inhaled Antibiotics in Bronchiectasis and Cystic Fibrosis Consortium, European Respiratory Society, and the British Lung Foundation.

TLR9 polymorphism correlates with immune activation, CD4 decline and plasma IP10 levels in HIV patients.

BACKGROUND: The mechanism behind HIV mediated immune activation remains debated, although the role of virus replication in this process is increasingly evident. Toll like Receptor 9 (TLR9) has been implicated in HIV mediated immune activation via sensing of viral CpG DNA. Polymorphisms in the TLR9 gene and promoter region including TLR9 1635A/G and 1486C/T have been found to be associated with multiple infectious diseases and cancers. METHODS: In the current study, we looked at the correlation of TLR9 polymorphisms 1635A/G and 1486C/T with key hallmarks of HIV disease in a cohort of 50 HIV infected patients. We analyzed CD4 counts, T cell immune activation characterized by upregulation of CD38 and HLA-DR and upregulation of plasma biomarkers of inflammation like LPS, sCD14, IL-6 and IP10 in the HIV patient cohort and compared it to healthy controls. RESULTS: We found that TLR9 1635AA genotype was associated with lower CD4 counts and significantly higher immune activation in both CD4+ and CD8+ T cells. Analysis of HIV associated plasma biomarkers including LPS, sCD14, IL-6 and IP10 revealed a strong correlation between IP10 and immune activation. Interestingly, IP10 levels were also found to be higher in HIV patients with the 1635AA genotype. Furthermore, the TLR9 1486C/T polymorphism that is in linkage disequilibrium with 1635A/G was weakly associated with lower CD4 counts, higher CD8 immune activation and higher IP10 levels. CONCLUSIONS: As TLR9 stimulation is known to induce IP10 production by dendritic cells, our findings provide new insights into HIV mediated immune activation and CD4 loss. TLR9 stimulation by viral CpG DNA may be important to HIV immunopathogenesis and the TLR9 polymorphisms 1635A/G and 1486C/T may be associated with disease progression.

HIV-1 Env Glycoprotein Phenotype along with Immune Activation Determines CD4 T Cell Loss in HIV Patients.

The mechanism behind the selective depletion of CD4(+) cells in HIV infections remains undetermined. Although HIV selectively infects CD4(+) cells, the relatively few infected cells in vivo cannot account for the extent of CD4(+) T cell depletion, suggesting indirect or bystander mechanisms. The role of virus replication, Env glycoprotein phenotype, and immune activation (IA) in this bystander phenomenon remains controversial. Using samples derived from HIV-infected patients, we demonstrate that, although IA in both CD4(+) and CD8(+) subsets correlates with CD4 decline, apoptosis in CD4(+) and not CD8(+) cells is associated with disease progression. Because HIV-1 Env glycoprotein has been implicated in bystander apoptosis, we cloned full-length Envs from plasma of viremic patients and tested their apoptosis-inducing potential (AIP). Interestingly, AIP of HIV-1 Env glycoproteins were found to correlate inversely with CD4:CD8 ratios, suggesting a role of Env phenotype in disease progression. In vitro mitogenic stimulation of PBMCs resulted in upregulation of IA markers but failed to alter the CD4:CD8 ratio. However, coculture of normal PBMCs with Env-expressing cells resulted in selective CD4 loss that was significantly enhanced by IA. Our study demonstrates that AIP of HIV-1 Env and IA collectively determine CD4 loss in HIV infection.

Identification of conserved motifs in the West Nile virus envelope essential for particle secretion.

BACKGROUND: Enveloped viruses utilize cellular membranes to bud from infected cells. The process of virion assembly and budding is often facilitated by the presence of certain conserved motifs within viral proteins in conjunction with cellular factors. We hence examined the West Nile Virus (WNV) Envelope protein for the presence of any such motifs and their functional characterization. RESULTS: We identified conserved 461PXAP464 and 349YCYL352 motifs in the WNV envelope glycoprotein bearing resemblance to retroviral late domains. Disruptive mutations of PXAP to LAAL and of the highly conserved Cys350 in the YCYL motif, led to a severe reduction in WNV particle production. Similar motifs in case of retroviruses are known to interact with components of host sorting machinery like PXAP with Tsg101 and YXXL with Alix. However, in the case of WNV, siRNA mediated depletion of Alix or Tsg101 did not have an effect on WNV release. Molecular modeling suggested that while the 461PXAP464 motif is surface accessible and could potentially interact with cellular proteins required for WNV assembly, the 349YCYL352 motif was found to be internal with Cys350 important for protein folding via disulphide bonding. CONCLUSIONS: The conserved 461PXAP464 and 349YCYL352 motifs in the WNV envelope are indispensable for WNV particle production. Although these motifs bear sequence similarity to retroviral late domains and are essential for WNV assembly, they are functionally distinct suggesting that they are not the typical late domain like motifs of retroviruses and may play a role other than Alix/Tsg101 utilization/dependence.

Synthesis and anti-tumor activity of carbohydrate analogues of the tetrahydrofuran containing acetogenins.

The tetrahydrofuran (THF) containing annonaceous acetogenins (AAs) are attractive candidates for drug development because of their potent cytotoxicity against a wide range of tumors and their relatively simple and robust structures. Replacement of the THF segment with a sugar residue may deliver analogues with improved tumor selectivity and pharmacokinetics and are therefore attractive for drug development. As a first test to the feasibility of such structures, a set of such monosaccharide analogues was synthesized and assayed against four human tumor cell lines, cervical (HeLa), breast (MDA-MB231), T-cell leukemia (Jurkat) and prostate (PC-3). Certain analogues showed low micromolar activity that was comparable to a structurally similar, naturally occurring mono-THF acetogenin. A preliminary examination of the structure-activity profile of these carbohydrate analogues suggests that they have a similar mechanism of action as their THF congeners.

TCR-induced T cell activation leads to simultaneous phosphorylation at Y505 and Y394 of p56(lck) residues.

Biochemical studies have demonstrated that phosphorylation of lymphocyte cell kinase (p56(lck) ) is crucial for activation of signaling cascades following T cell receptor (TCR) stimulation. However, whether phosphorylation/dephosphorylation of the activating or inhibitory tyrosine residues occurs upon activation is controversial. Recent advances in intracellular staining of phospho-epitopes and cytometric analysis, requiring few cells, have opened up novel avenues for the field of immunological signaling. Here, we assessed p56(lck) phosphorylation, using a multiparameter flow-cytometric based detection method following T cell stimulation. Fixation and permeabilization in conjunction with zenon labeling technology and/or fluorescently labeled antibodies against total p56(lck) or cognate phospho-tyrosine (pY) residues or surface receptors were used for detection purposes. Our observations showed that activation of Jurkat or primary human T cells using H(2) O(2) or TCR-induced stimulation led to simultaneous phosphorylation of the activating tyrosine residue, Y394 and the inhibitory tyrosine residue, Y505 of p56(lck) . This was followed by downstream calcium flux and expression of T cell activation markers; CD69 and CD40 ligand (CD40L). However, the extent of measurable activation readouts depended on the optimal stimulatory conditions (temperature and/or stimuli combinations). Treatment of cells with a p56(lck) -specific inhibitor, PP2, abolished phosphorylation at either residue in a dose-dependent manner. Taken together, these observations show that TCR-induced stimulation of T cells led to simultaneous phosphorylation of p56(lck) residues. This implies that dephosphorylation of Y505 is not crucial for p56(lck) activity. Also, it is clear that cytometric analysis provides for a rapid, sensitive, and quantitative method to supplement biochemical studies on p56(lck) signaling pathways in T cells at single cell level.

Synthesis, gp120 binding and anti-HIV activity of fatty acid esters of 1,1-linked disaccharides.

Inspired by the anti-human immunodeficiency virus (HIV) activity of analogues of ß-galactosylceramide (GalCer), a set of mono- and di-saccharide fatty acid esters were designed as GalCer mimetics and their binding to the V3 loop peptide of HIV-1 and anti-HIV activity evaluated. 1,1-linked Gal-Man and Glu-Man disaccharides with an ester on the Man subunit bound the V3 loop peptide and inhibited HIV infectivity in single round infection assays with the TZM-bl cell line. IC(50)'s were in the 50 µM range with no toxicity to the cells at concentrations up to 200 µM. These compounds appear to inhibit virus entry at early steps in viral infection since they were inactive if added post viral entry. Although these compounds were found to bind to the V3 loop peptide of gp120, it is not clear that this interaction is responsible for their anti-HIV activity because the relative binding affinity of closely related analogues did not correlate with their antiviral behavior. The low cytotoxicity of these 1,1-linked disaccharide fatty acid esters, combined with the easy accessibility to structurally diverse analogues make these molecules attractive leads for new topical anti-viral agents.

Targeting the HIV entry, assembly and release pathways for anti-HIV gene therapy.

Targeting the HIV entry and assembly pathways holds promise for development of novel anti-HIV gene therapy vectors. We characterized discrete dominant negative (DN) Gag and Envelope mutants for their anti-HIV-1 activity. We show here that capsid mutants (Q155N and Y164A) are more potent inhibitors of WT HIV than the matrix mutant 1GA. Both the Envelope mutants tested, V513E and R515A, were equally effective and a combination of Gag and Envelope DN genes significantly enhanced potency. Interestingly, the DN mutants acted at multiple steps in the virus life cycle rather than solely disrupting virus release or infection. Inhibition mediated by R515A could be partially attributed to the Envelope cytoplasmic tail, as deletion of R515A tail partially abrogated its DN effect. Finally, the Y164A/R515A double mutant expressed in a lentiviral vector was effective at inhibiting HIV replication in CD34+ hematopoietic stem cell-derived macrophages, demonstrating the therapeutic potential of our approach.

Specific targeting to B cells by lipid-based nanoparticles conjugated with a novel CD22-ScFv.

The CD22 antigen is a viable target for therapeutic intervention for B-cell lymphomas. Several therapeutic anti-CD22 antibodies as well as an anti-CD22-based immunotoxin (HA22) are currently under investigation in clinical settings. Coupling of anti-CD22 reagents with a nano-drug delivery vehicle is projected to significantly improve treatment efficacies. Therefore, we generated a mutant of the targeting segment of HA22 (a CD22 scFv) to increase its soluble expression (mut-HA22), and conjugated it to the surface of sonicated liposomes to generate immunoliposomes (mut-HA22-liposomes). We examined liposome binding and uptake by CD22(+) B-lymphocytes (BJAB) by using calcein and/or rhodamine PE-labeled liposomes. We also tested the effect of targeting on cellular toxicity with doxorubicin-loaded liposomes. We report that: (i) Binding of mut-HA22-liposomes to BJAB cells was significantly greater than liposomes not conjugated with mut-HA22 (control liposomes), and mut-HA22-liposomes bind to and are taken in by BJAB cells in a dose and temperature-dependent manner, respectively; (ii) This binding occurred via the interaction with the cellular CD22 as pre-incubation of the cells with mut-HA22 blocked subsequent liposome binding; (iii) Intracellular localization of mut-HA22-liposomes at 37 degrees C but not at 4 degrees C indicated that our targeted liposomes were taken up through an energy dependent process via receptor-mediated endocytosis; and (iv) Mut-HA22-liposomes loaded with doxorubicin exhibited at least 2-3 fold more accumulation of doxorubicin in BJAB cells as compared to control liposomes. Moreover, these liposomes showed at least a 2-4 fold enhanced killing of BJAB or Raji cells (CD22(+)), but not SUP-T1 cells (CD22(-)). Taken together these data suggest that these 2nd-generation liposomes may serve as promising carriers for targeted drug delivery to treat patients suffering from B-cell lymphoma.

Photo-activation of the hydrophobic probe iodonaphthylazide in cells alters membrane protein function leading to cell death.

BACKGROUND: Photo-activation of the hydrophobic membrane probe 1, 5 iodonaphthylazide (INA) by irradiation with UV light (310-380 nm) results in the covalent modification of transmembrane anchors of membrane proteins. This unique selectivity of INA towards the transmembrane anchor has been exploited to specifically label proteins inserted in membranes. Previously, we have demonstrated that photo-activation of INA in enveloped viruses resulted in the inhibition of viral membrane protein-induced membrane fusion and viral entry into cells. In this study we show that photo-activation of INA in various cell lines, including those over-expressing the multi-drug resistance transporters MRP1 or Pgp, leads to cell death. We analyzed mechanisms of cell killing by INA-UV treatment. The effects of INA-UV treatment on signaling via various cell surface receptors, on the activity of the multi-drug resistance transporter MRP1 and on membrane protein lateral mobility were also investigated. RESULTS: INA treatment of various cell lines followed by irradiation with UV light (310-380 nm) resulted in loss of cell viability in a dose dependent manner. The mechanism of cell death appeared to be apoptosis as indicated by phosphatidylserine exposure, mitochondrial depolarization and DNA fragmentation. Inhibition by pan-caspase inhibitors and cleavage of caspase specific substrates indicated that at low concentrations of INA apoptosis was caspase dependent. The INA-UV treatment showed similar cell killing efficacy in cells over-expressing MRP1 function as control cells. Efflux of an MRP1 substrate was blocked by INA-UV treatment of the MRP1-overexpressing cells. Although INA-UV treatment resulted in inhibition of calcium mobilization triggered by chemokine receptor signaling, Akt phosphorylation triggered by IGF1 receptor signaling was enhanced. Furthermore, fluorescence recovery after photobleaching experiments indicated that INA-UV treatment resulted in reduced lateral mobility of a seven transmembrane G protein-coupled receptor. CONCLUSION: INA is a photo-activable agent that induces apoptosis in various cancer cell lines. It reacts with membrane proteins to alter the normal physiological function resulting in apoptosis. This activity of INA maybe exploited for use as an anti-cancer agent.

Ceramides: branched alkyl chains in the sphingolipid siblings of diacylglycerol improve biological potency.

The synthesis of a small number of ceramide analogues containing a combination of linear and highly branched alkyl chains on either the d-sphingosine or the N-acyl core of the molecule is reported. Regardless of location, the presence of the branched chain improves potency relative to the positive control, C2 ceramide; however, the most potent compound (4) has the branched side chain as part of the d-sphingosine core. The induction of apoptosis by 4 in terms of Annexin V binding and DiOC(6) labeling was superior to that achieved with C2 ceramide.

Site-specific mutations in HIV-1 gp41 reveal a correlation between HIV-1-mediated bystander apoptosis and fusion/hemifusion.

The loss of CD4(+) T cells in HIV-1 infections is hypothesized to be caused by apoptosis of bystander cells mediated by cell surface-expressed HIV-1 Env glycoprotein. However, the mechanism by which Env mediates this process remains controversial. Specifically, the role of HIV-1 gp120 binding to CD4 and CXCR4 versus the fusion process mediated by gp41 remains unresolved. Env-induced apoptosis in bystander cells has been shown to be gp41-dependent and correlates with the redistribution of membrane lipids between Env-expressing cells and target cells (hemifusion). Using a rational mutagenesis approach aimed at targeting Env function via the gp41 subunit, we examined the role of HIV gp41 in bystander apoptosis. A mutation in the fusion domain of gp41 (V513E) resulted in a fusion-defective Env that failed to induce apoptosis. A mutation in the gp41 N-terminal helix (G547D) reduced cell fusion capacity and apoptosis; conversely, an Env mutant with a deletion of the gp41 cytoplasmic tail (Ct Del) enhanced both cell-to-cell fusion and apoptosis. Most significantly, an Env mutant containing a substitution in the loop region of gp41 (D589L) mediated transfer of lipids (hemifusion) to bystander cells but was defective in cell-to-cell and to a lesser degree virus-to-cell fusion. This mutant was still able to induce apoptosis in bystander cells. Hence, we have provided the first direct evidence that gp41-mediated hemifusion is both required and sufficient for induction of apoptosis in bystander cells. These results may help to explain the mechanism of HIV-1 Env-induced T cell depletion.

Feline immunodeficiency virus envelope glycoprotein mediates apoptosis in activated PBMC by a mechanism dependent on gp41 function.

Feline Immunodeficiency Virus (FIV) is a lentivirus that causes immunodeficiency in cats, which parallels HIV-1-induced immunodeficiency in humans. It has been established that HIV envelope (Env) glycoprotein mediates T cell loss via a mechanism that requires CXCR4 binding. The Env glycoprotein of FIV, similar to HIV, requires CXCR4 binding for viral entry, as well as inducing membrane fusion leading to syncytia formation. However, the role of FIV Env in T cell loss and the molecular mechanisms governing this process have not been elucidated. We studied the role of Env glycoprotein in FIV-mediated T cell apoptosis in an in vitro model. Our studies demonstrate that membrane-expressed FIV Env induces apoptosis in activated feline peripheral blood mononuclear cells (PBMC) by a mechanism that requires CXCR4 binding, as the process was inhibited by CXCR4 antagonist AMD3100 in a dose-dependent manner. Interestingly, studies regarding the role of CD134, the recently identified primary receptor of FIV, suggest that binding to CD134 may not be important for induction of apoptosis in PBMC. However, inhibiting Env-mediated fusion post CXCR4 binding by FIV gp41-specific fusion inhibitor also inhibited apoptosis. Under similar conditions, a fusion-defective gp41 mutant was unable to induce apoptosis in activated PBMC. Our findings are the first report suggesting the potential of FIV Env to mediate apoptosis in bystander cells by a process that is dependent on gp41 function.