Expression of the extracellular domain of the human heat-stable enterotoxin receptor in Escherichia coli and generation of neutralizing antibodies

The entire extracellular domain of the human heat-stable enterotoxin (ST) receptor as well as a truncated N-terminal domain were cloned as glutathione S-transferase fusion proteins and expressed in Escherichia coli. The recombinant fusion proteins were purified from both the cytosol and the inclusion body fractions by selective detergent extraction followed by glutathione-agarose affinity chromatography. The purified protein, corresponding to the entire extracellular domain, bound the stable toxin peptide with an affinity comparable to that of the native receptor characterized from the human colonic T84 cell line. No binding was observed with the N-terminal truncated fragment of the receptor under similar conditions, Polyclonal antibodies were raised to the entire extracellular domain fusion protein as well as the truncated extracellular domain fusion protein, and the antibodies were purified by affinity chromatography. Addition of the purified antibodies to T84 cells inhibited ST binding and abolished ST-mediated cGMP production, indicating that critical epitopes involved in ligand interaction are present in the N-terminal fragment of the receptor, Purified antibodies recognized a single protein of M(r) 160,000 Da on Western blotting with T84 membranes, corresponding to a size of the native glycosylated receptor in T84 cells. These studies are the first report of the expression, purification, and characterization of any member of the guanylyl cyclase family of receptors in E. coli and show that binding of the toxin to the extracellular domain of the receptor is possible in the absence of any posttranslational modifications such as glycosylation. The recombinant fusion proteins as well as the antibodies that we have generated could serve as useful tools in the identification of critical residues of the extracellular domain involved in ligand interaction.

Hyperexpression of biologically active human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris

Human chorionic gonadotropin (hCG), a heterodimeric glycoprotein hormone, is composed of an alpha subunit noncovalentlv associated with the hormone-specific beta subunit. The objective of the present study was recombinant expression of properly folded, biologically active hCG and its subunits using an expression system that could be used for structure-function studies while providing adequate quantities of the hormone for immunocontraceptive studies. We report here expression of biologically active hCG and its subunits using a yeast expression system, Pichia pastoris. The recombinant hGG alpha and hCG beta subunits were secreted into the medium and the levels of expression achieved at shake culture level were 24 and 2.7-3 mg/l secretory medium respectively. Go-expression of both subunits in the same cell resulted in secretion of heterodimeric hGG into the medium. The pichia-expressed hCG was immunologically similar to the native hormone, capable of binding to the LH receptors and stimulating a biological response in vitro. Surprisingly, the maximal response obtained was twice that obtained with the native hGG. The le level of expression of hCG achieved was 12-16 mg/l secretory medium and is expected to increase several-fold in a fermenter. Thus the Pichia expression system is capable of hyperexpressing properly folded, biologically active hGG and is suitable for structure-function studies of the hormone.

Localisation of Plasmodium falciparum uroporphyrinogen III decarboxylase of the heme-biosynthetic pathway in the apicoplast and characterisation of its catalytic properties

Uroporphyrinogen decarboxylase (UROD) is a key enzyme in the heme-biosynthetic pathway and in Plasmodium falciparum it occupies a strategic position in the proposed hybrid pathway for heme biosynthesis involving shuttling of intermediates between different subcellular compartments in the parasite. In the present study, we demonstrate that an N-terminally truncated recombinant P. falciparum UROD (r(Δ)PfUROD) over-expressed and purified from Escherichia coli cells, as well as the native enzyme from the parasite were catalytically less efficient compared with the host enzyme, although they were similar in other enzyme parameters. Molecular modeling of PfUROD based on the known crystal structure of the human enzyme indicated that the protein manifests a distorted triose phosphate isomerase (TIM) barrel fold which is conserved in all the known structures of UROD. The parasite enzyme shares all the conserved or invariant amino acid residues at the active and substrate binding sites, but is rich in lysine residues compared with the host enzyme. Mutation of specific lysine residues corresponding to residues at the dimer interface in human UROD enhanced the catalytic efficiency of the enzyme and dimer stability indicating that the lysine rich nature and weak dimer interface of the wild-type PfUROD could be responsible for its low catalytic efficiency. PfUROD was localised to the apicoplast, indicating the requirement of additional mechanisms for transport of the product coproporphyrinogen to other subcellular sites for its further conversion and ultimate heme formation.

Assessment of luteal rescue and desensitization of macaque corpus luteum brought about by human chorionic gonadotrophin and deglycosylated human chorionic gonadotrophin treatment

The objective of the current study was to investigate the mechanism by which the corpus luteum (CL) of the monkey undergoes desensitization to luteinizing hormone following exposure to increasing concentration of human chorionic gonadotrophin (hCG) as it occurs in pregnancy. Female bonnet monkeys were injected (im) increasing doses of hCG or dghCG beginning from day 6 or 12 of the luteal phase for either 10 or 4 or 2 days. The day of oestrogen surge was considered as day '0' of luteal phase. Luteal cells obtained from CL of these animals were incubated with hCG (2 and 200 pg/ml) or dbcAMP (2.5, 25 and 100 mu M) for 3 h at 37 degrees C and progesterone secreted was estimated. Corpora lutea of normal cycling monkeys on day 10/16/22 of the luteal phase were used as controls, In addition the in vivo response to CG and deglycosylated hCG (dghCG) was assessed by determining serum steroid profiles following their administration. hCG (from 15-90 IU) but not dghCG (15-90 IU) treatment in vivo significantly (P < 0.05) elevated serum progesterone and oestradiol levels. Serum progesterone, however, could not be maintained at a elevated level by continuous treatment with hCG (from day 6-15), the progesterone level declining beyond day 13 of luteal phase. Administering low doses of hCG (15-90 IU/day) from day 6-9 or high doses (600 IU/day) on days 8 and 9 of the luteal phase resulted in significant increase (about 10-fold over corresponding control P < 0.005) in the ability of luteal cells to synthesize progesterone (incubated controls) in vitro. The luteal cells of the treated animals responded to dbcAMP (P < 0.05) but not to hCG added in vitro, The in vitro response of luteal cells to added hCG was inhibited by 0, 50 and 100% if the animals were injected with low (15-90 IU) or medium (100 IU) between day 6-9 of luteal phase and high (600 IU on day 8 and 9 of luteal phase) doses of dghCG respectively; such treatment had no effect on responsivity of the cells to dbcAMP, The luteal cell responsiveness to dbcAMP in vitro was also blocked if hCG was administered for 10 days beginning day 6 of the luteal phase. Though short term hCG treatment during late luteal phase (from days 12-15) had no effect on luteal function, 10 day treatment beginning day 12 of luteal phase resulted in regain of in vitro responsiveness to both hCG (P < 0.05) and dbcAMP (P < 0.05) suggesting that luteal rescue can occur even at this late stage. In conclusion, desensitization of the CL to hCG appears to be governed by the dose/period for which it is exposed to hCG/dghCG. That desensitization is due to receptor occupancy is brought out by the fact that (i) this can be achieved by giving a larger dose of hCG over a 2 day period instead of a lower dose of the hormone for a longer (4 to 10 days) period and (ii) the effect can largely be reproduced by using dghCG instead of hCG to block the receptor sites. It appears that to achieve desensitization to dbcAMP also it is necessary to expose the luteal cell to relatively high dose of hCG for more than 4 days.

Molecule Modeling of Human Pentameric alpha(7) Neuronal Nicotinic Acetylcholine Receptor and Its Interaction with its Agonist and Competitive Antagonist

The nicotinic Acetylcholine Receptor (nAChR) is the major class of neurotransmitter receptors that is involved in many neurodegenerative conditions such as schizophrenia, Alzheimer's and Parkinson's diseases. The N-terminal region or Ligand Binding Domain (LBD) of nAChR is located at pre- and post-synaptic nervous system, which mediates synaptic transmission. nAChR acts as the drug target for agonist and competitive antagonist molecules that modulate signal transmission at the nerve terminals. Based on Acetylcholine Binding Protein (AChBP) from Lymnea stagnalis as the structural template, the homology modeling approach was carried out to build three dimensional model of the N-terminal region of human alpha(7)nAChR. This theoretical model is an assembly of five alpha(7) subunits with 5 fold axis symmetry, constituting a channel, with the binding picket present at the interface region of the subunits. alpha-netlrotoxin is a potent nAChR competitive antagonist that readily blocks the channel resulting in paralysis. The molecular interaction of alpha-Bungarotoxin, a long chain alpha-neurotoxin from (Bungarus multicinctus) and human alpha(7)nAChR seas studied. Agonists such as acetylcholine, nicotine, which are used in it diverse array of biological activities, such as enhancements of cognitive performances, were also docked with the theoretical model of human alpha(7)nAChR. These docked complexes were analyzed further for identifying the crucial residues involved i interaction. These results provide the details of interaction of agonists and competitive antagonists with three dimensional model of the N-terminal region of human alpha(7)nAChR and thereby point to the design of novel lead compounds.

Nutritional rehabilitation in cystic fibrosis: A 5 year follow-up study

Previously, we reported catch-up weight gain, growth, and improved lung function in a group of malnourished cystic fibrosis (CF) children receiving aggressive nutritional supplementation for 1 year compared with a forced expiratory volume in 1 s (FEV1)-, height-, and sex-matched comparison group receiving standard therapy. To evaluate long-term effects, the clinical progress of both groups has been studied over a 5 year period. The supplemented group (n = 10) received supplements for a median of 1.35 years to achieve nutritional rehabilitation. Compared with the nonsupplemented group (n = 14), the previously supplemented group had lower mortality (2 vs. 4, N.S.) and significantly greater weight and height z scores at 4 and 5 years. The progression of pulmonary function abnormalities as measured by FEV, and forced vital capacity (FVC) slopes was greater at 3 years in the nonsupplemented group (FEV1, p < 0.05) but no significant differences in rates of deterioration of pulmonary function were seen after 5 years in the two groups of survivors. We conclude that intensive nutritional support for 1 year has both short-and long-term effects on nutrition and growth, still evident some years after the cessation of this therapeutic modality. Supplementation for periods of longer than 1 year may produce greater gains and possibly prolong the improvement in pulmonary function observed in the earlier study. © 1992 Raven Press, Ltd., New York.

Temporal dependence of in vivo USPIO-enhanced MRI signal changes in human carotid atheromatous plaques

Introduction: Ultrasmall superparamagnetic iron oxide (USPIO)-enhanced MRI has been shown to be a useful modality to image activated macrophages in vivo, which are principally responsible for plaque inflammation. This study determined the optimum imaging time-window to detect maximal signal change post-USPIO infusion using T1-weighted (T1w), T2*- weighted (T2*w) and quantitative T2*(qT 2*) imaging. Methods: Six patients with an asymptomatic carotid stenosis underwent high resolution T1w, T2*w and qT2*MR imaging of their carotid arteries at 1.5 T. Imaging was performed before and at 24, 36, 48, 72 and 96 h after USPIO (Sinerem™, Guerbet, France) infusion. Each slice showing atherosclerotic plaque was manually segmented into quadrants and signal changes in each quadrant were fitted to an exponential power function to model the optimum time for post-infusion imaging. Results: The power function determining the mean time to convergence for all patients was 46, 41 and 39 h for the T1w, T 2*w and qT2*sequences, respectively. When modelling each patient individually, 90% of the maximum signal intensity change was observed at 36 h for three, four and six patients on T1w, T 2*w and qT2*, respectively. The rates of signal change decrease after this period but signal change was still evident up to 96 h. Conclusion: This study showed that a suitable imaging window for T 1w, T2*w and qT2*signal changes post-USPIO infusion was between 36 and 48 h. Logistically, this would be convenient in bringing patients back for one post-contrast MRI, but validation is required in a larger cohort of patients.

Survival, growth and quality of life in children after orthotopic liver transplantation: A 5 year experience

The aims of this study were to investigate outcome and to evaluate areas of potential ongoing concern after orthotopic liver transplantation (OLT) in children. Actuarial survival in relation to age and degree of undernutrition at the time of OLT was evaluated in 53 children (age 0.58-14.2 years) undergoing OLT for endstage liver disease. Follow-up studies of growth and quality of life were undertaken in those with a minimum follow-up period of 12 months (n = 26). The overall 3 year actuarial survival was 70%. Survival rates did not differ between age groups (actuarial 2 year survival for ages <1, 1-5 and >5 years were 70, 70 and 69% respectively) but did differ according to nutritional status at OLT (actuarial 2 year survival for children with Z scores for weight <-1 was 57%, >-1 was 95%; P = 0.004). Significant catch-up weight gain was observed by 18 months post-transplant, while height improved less rapidly. Quality of life (assessed by Vineland Adaptive Behaviour Scales incorporating socialization, daily living skills, communication and motor skills) was good (mean composite score 91 ± 19). All school-aged children except one were attending normal school. Two children had mild to moderate intellectual handicap related to post-operative intracerebral complications. Satisfactory long-term survival can be achieved after OLT in children regardless of age but the importance of pre-operative nutrition is emphasized. Survivors have an excellent chance of a good quality of life and of satisfactory catch-up weight gain and growth.

The ontogeny of serum immunoreactive pancreatic lipase and cationic trypsinogen in the premature human infant

We evaluated the development of the exocrine pancreas in 16 healthy preterm infants (29.3 ± 1.6 weeks). The infants were fed breast milk with formula supplements (n=8) or formula alone (n=8). Growth was monitored weekly for 12 weeks then at 3, 6, 9, 12 months. At the same intervals sera were determined for pancreatic lipase and cationic trypsinogen. In addition, cord blood samples were analysed from another 33 preterm (27.6 ± 5.2 weeks) and 75 healthy full-term infants. Serum pancreatic lipase in the cord blood of term (3.7 ± 0.4 μg/l) and preterm infants (1.8 ± 0.2 μg/l) was significantly below values reported for older children (10.5 ± 0.9 μg/l; p < 0.001). In the preterm infant, serum lipase was also significantly lower than values obtained at term (p < 0.001). At birth, serum trypsinogen for preterm (16.8 ± 1.3 μg/l) and term infants (23.3 ± 1.9 μg/l) were below those for older children (31.4 ± 3.7 μg/l; p < 0.05). Over the first 3 weeks of life, serum lipase and trypsinogen increased significantly. From 3 weeks to 12 months of age, serum trypsinogen values remained unchanged, but serum lipase increased dramatically after 10 weeks of age. Thus, at 6 and 12 months of age, the preterm infants had significantly higher serum lipase values than infants of the same age born at term. These two pancreatic enzymes appear to show independent age-related maturation in infants born before term. The rate of maturation of lipase appears to be accelerated by exposure to the extrauterine environment.

GABAAα1 and GABAAρ1 subunits are expressed in cultured human RPE cells and GABAA receptor agents modify the intracellular calcium concentration.

Purpose: Gamma-aminobutyric acid A (GABAA) receptors (GABAARs), which are ionotropic receptors involving chloride channels, have been identified in various neural (e.g., mouse retinal ganglion cells) and nonneural cells (e.g., mouse lens epithelial cells) regulating the intracellular calcium concentration ([Ca(2+)]i). GABAAR β-subunit protein has been isolated in the cultured human and rat RPE, and GABAAα1 and GABAAρ1 mRNAs and proteins are present in the chick RPE. The purpose of this study was to investigate the expression of GABAAα1 and GABAAρ1, two important subunits in forming functional GABAARs, in the cultured human RPE, and further to explore whether altering receptor activation modifies [Ca(2+)]i. Methods: Human RPE cells were separately cultured from five donor eye cups. Real-time PCR, western blots, and immunofluorescence were used to test for GABAAα1 and GABAAρ1 mRNAs and proteins. The effects of the GABAAR agonist muscimol, antagonist picrotoxin, or the specific GABAAρ antagonist 1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA) on [Ca(2+)]i in cultured human RPE were demonstrated using Fluo3-AM. Results: Both GABAAα1 and GABAAρ1 mRNAs and proteins were identified in cultured human RPE cells; antibody staining was mainly localized to the cell membrane and was also present in the cytoplasm but not in the nucleus. Muscimol (100 μM) caused a transient increase of the [Ca(2+)]i in RPE cells regardless of whether Ca(2+) was added to the buffer. Muscimol-induced increases in the [Ca(2+)]i were inhibited by pretreatment with picrotoxin (300 μM) or TPMPA (500 μM). Conclusions: GABAAα1 and GABAAρ1 are expressed in cultured human RPE cells, and GABAA agents can modify [Ca(2+)]i.

Social perception and cognition : processing of gestures, postures and facial expressions in the human brain

Humans are a social species with the internal capability to process social information from other humans. To understand others behavior and to react accordingly, it is necessary to infer their internal states, emotions and aims, which are conveyed by subtle nonverbal bodily cues such as postures, gestures, and facial expressions. This thesis investigates the brain functions underlying the processing of such social information. Studies I and II of this thesis explore the neural basis of perceiving pain from another person s facial expressions by means of functional magnetic resonance imaging (fMRI) and magnetoencephalography (MEG). In Study I, observing another s facial expression of pain activated the affective pain system (previously associated with self-experienced pain) in accordance with the intensity of the observed expression. The strength of the response in anterior insula was also linked to the observer s empathic abilities. The cortical processing of facial pain expressions advanced from the visual to temporal-lobe areas at similar latencies (around 300 500 ms) to those previously shown for emotional expressions such as fear or disgust. Study III shows that perceiving a yawning face is associated with middle and posterior STS activity, and the contagiousness of a yawn correlates negatively with amygdalar activity. Study IV explored the brain correlates of interpreting social interaction between two members of the same species, in this case human and canine. Observing interaction engaged brain activity in very similar manner for both species. Moreover, the body and object sensitive brain areas of dog experts differentiated interaction from noninteraction in both humans and dogs whereas in the control subjects, similar differentiation occurred only for humans. Finally, Study V shows the engagement of the brain area associated with biological motion when exposed to the sounds produced by a single human being walking. However, more complex pattern of activation, with the walking sounds of several persons, suggests that as the social situation becomes more complex so does the brain response. Taken together, these studies demonstrate the roles of distinct cortical and subcortical brain regions in the perception and sharing of others internal states via facial and bodily gestures, and the connection of brain responses to behavioral attributes.

Development of a vaccine for Chlamydia trachomatis: Challenges and current progress

Chlamydia trachomatis remains an enigmatic bacterial pathogen with no vaccine yet available to treat human ocular and genital tract infections caused by tissue-tropic serovars of the organism. Globally, it is the leading cause of preventable blindness as well as the leading cause of bacterial sexually transmitted infections. The pathogen has a range of virulence factors that enable it to successfully evade both the innate and adaptive immune system of the host. The host immune system, although protective, paradoxically is also associated closely with the pathologies of trachoma and pelvic inflammatory disease – disease sequelae of some chlamydial infections and reinfections in some genetically susceptible hosts. In this review, we focus on what is known currently about the pathogenesis of ocular and genital infections caused by this mucosal pathogen. We also discuss novel insights into the pathogenesis of infections caused by the genital and ocular serovars of C. trachomatis, including a discussion of both pathogen and host factors, such as the human microbiota at these mucosal sites as well as the current immunological challenges facing vaccine development. Finally, we discuss the current progress toward development of a vaccine against C. trachomatis. A wide range of recombinant protein antigens are being identified and, hence, are available for vaccine trials. A plasmid-free live strain has recently been produced and evaluated in the mouse (Chlamydia muridarum) and monkey (C. trachomatis) models. The data for ocular infections in the monkey model was particularly encouraging, although the path to regulatory approval of a live vaccine is still uncertain. While still a major challenge, vaccines for ocular and genital C. trachomatis infections are looking more promising.

Adenoviral Gene Therapy for Advanced Head and Neck Cancer

Advanced stage head and neck cancers (HNC) with distant metastasis, as well as prostate cancers (PC), are devastating diseases currently lacking efficient treatment options. One promising developmental approach in cancer treatment is the use of oncolytic adenoviruses, especially in combination therapy with conventional cancer therapies. The safety of the approach has been tested in many clinical trials. However, antitumor efficacy needs to be improved in order to establish oncolytic viruses as a viable treatment alternative. To be able to test in vivo the effects on anti-tumor efficiency of a multimodal combination therapy of oncolytic adenoviruses with the standard therapeutic combination of radiotherapy, chemotherapy and Cetuximab monoclonal antibody (mAb), a xenograft HNC tumor model was developed. This model mimics the typical clinical situation as it is initially sensitive to cetuximab, but resistance develops eventually. Surprisingly, but in agreement with recent findings for chemotherapy and radiotherapy, a higher proportion of cells positive for HNC cancer stem cell markers were found in the tumors refractory to cetuximab. In vitro as well as in vivo results found in this study support the multimodal combination therapy of oncolytic adenoviruses with chemotherapy, radiotherapy and monoclonal antibody therapy to achieve increased anti-tumor efficiency and even complete tumor eradication with lower treatment doses required. In this study, it was found that capsid modified oncolytic viruses have increased gene transfer to cancer cells as well as an increased antitumor effect. In order to elucidate the mechanism of how oncolytic viruses promote radiosensitization of tumor cells in vivo, replicative deficient viruses expressing several promising radiosensitizing viral proteins were tested. The results of this study indicated that oncolytic adenoviruses promote radiosensitization by delaying the repair of DNA double strand breaks in tumor cells. Based on the promising data of the first study, two tumor double-targeted oncolytic adenoviruses armed with the fusion suicide gene FCU1 or with a fully human mAb specific for human Cytotoxic T Lymphocyte-Associated Antigen 4 (CTLA-4) were produced. FCU1 encodes a bifunctional fusion protein that efficiently catalyzes the direct conversion of 5-FC, a relatively nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine monophosphate, bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. Anti-CTLA4 mAb promotes direct killing of tumor cells via apoptosis and most importantly immune system activation against the tumors. These armed oncolytic viruses present increased anti-tumor efficacy both in vitro and in vivo. Furthermore, by taking advantage of the unique tumor targeted gene transfer of oncolytic adenoviruses, functional high tumor titers but low systemic concentrations of the armed proteins were generated. In addition, supernatants of tumor cells infected with Ad5/3-24aCTLA4, which contain anti-CTLA4 mAb, were able to effectively immunomodulate peripheral blood mononuclear cells (PBMC) of cancer patients with advanced tumors. -- In conclusion, the results presented in this thesis suggest that genetically engineered oncolytic adenoviruses have great potential in the treatment of advanced and metastatic HNC and PC.

Expression of cytochrome P450 enzymes and metabolism of timolol in human ocular tissue

Glaucoma is a group of progressive optic neuropathies causing irreversible blindness if not diagnosed and treated in the early state of progression. Disease is often, but not always, associated with increased intraocular pressure (IOP), which is also the most important risk factor for glaucoma. Ophthlamic timolol preparations have been used for decades to lower increased intraocular pressure (IOP). Timolol is locally well tolerated but may cause e.g. cardiovascular and pulmonary adverse effects due to systemic absorption. It has been reported that approximately 80% of a topically administered eye drop is systemically absorbed. However, only limited information is available on timolol metabolism in the liver or especially in the human eye. The aim of this work was to investigate metabolism of timolol in human liver and human ocular tissues. The expression of drug metabolizing cytochrome P450 (CYP) enzymes in the human ciliary epithelial cells was studied. The metabolism of timolol and the interaction potential of timolol with other commercially available medicines were investigated in vitro using different liver preparations. The absorption of timolol to the aqueous humor from two commercially available products: 0.1% eye gel and 0.5% eye drops and the presence of timolol metabolites in the aqueous humor were investigated in a clinical trial. Timolol was confirmed to be metabolized mainly by CYP2D6 as previously suggested. Potent CYP2D6 inhibitors especially fluoxetine, paroxetine and quinidine inhibited the metabolism of timolol. The inhibition may be of clinical significance in patients using ophthalmic timolol products. CYP1A1 and CYP1B1 mRNAs were expressed in the human ciliary epithelial cells. CYP1B1 was also expressed at protein level and the expression was strongly induced by a known potent CYP1B1 inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The CYP1B1 induction is suggested to be mediated by aryl hydrocarbon receptor (AHR). Low levels of CYP2D6 mRNA splice variants were expressed in the human ciliary epithelial cells and very low levels of timolol metabolites were detected in the human aqueous humor. It seems that negligible amount of CYP2D6 protein is expressed in the human ocular tissues. Timolol 0.1% eye gel leads to aqueous humor concentration high enough to achieve therapeutic effect. Inter-individual variation in concentrations is low and intraocular as well as systemic safety can be increased when using this product with lower timolol concentration instead of timolol 0.5% eye drops.