Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Introduction: Chronic HIV-1 infection is characterized by immune activation and dysregulation of immune homeostasis, which impacts on multiple immune cell types. The B-cell compartment, which plays an important role in the producing neutralizing antibodies, is also dysregulated in HIV- 1 infection. In this study we investigated peripheral blood B-cell subset distribution, and changes in expression of cellular activation, inhibition, and apoptosis signaling markers in both untreated chronic HIV-1 infected individuals and healthy uninfected controls. The neuropeptide immune modulator, vasoactive intestinal peptide (VIP) is known to selectively down-regulate activation of CD4+ T-cells in various disease settings including HIV-1, however to our knowledge, no studies have investigated the effect of VIP inhibition on B-cell activation. Materials & Methods: A total of 21 HIV+ve (CD4 count >250 cells/µl), and 19 HIV-ve individuals were recruited from the Emavundleni voluntary testing and counseling clinic in Crossroads, Western Province, South Africa. Whole blood was stained to distinguish B-cell subsets (activated memory (AM: CD21-CD27+), resting memory (RM: CD21+CD27+), mature naïve (MN: CD21+CD27-), or tissue-like memory (TLM: CD21loCD27lo). In addition expression of markers of B-cell activation (CD126, CD86, CD38, CD284, CD287), inhibition (CD72, CD85j, CD300a, CD305, CD307d), and apoptosis signaling (CD95), was assessed ex vivo by flow cytometry (BD FACSCanto II). For determination of functional responsiveness isolated B-cells (RosetteSep, Stemcell Technologies) were cultured for 18h (37°C, 5%CO2) without stimulation or stimulated with TLR ligands (LPS or R848). Stimulation experiments were also performed in the presence or absence of VIP. Results: Chronic HIV-1 infection affected B-cell subset distribution. The percentage (%) of TLM was increased by 59.24%, and %RM was decreased by 22.73% (both p<0.01). Total expression of the VIP receptor VPAC2 was decreased by 47.35% (p=0.0296). Subsets had a mixed phenotype ex vivo; HIV infection upregulated CD38 (by 59.56%, p=0.0004), CD72 (by 60.70%, p=0.0396), CD307d (by 68.63%, p=0.0015) on AM, while RM B-cells had increased expression of TLR4 (by 107.04%, p=0.0057) and TLR7 (by 208.14%, p=0.0199). TLM B cells (i.e. exhausted phenotype) displayed upregulated TLR7 (by 550%, p=0.0128) and CD307d (by 72.40% p=0.045) expression. MN B-cells had increased CD72 expression (by 70.98%, p=0.0026). R848 upregulated CD86 expression by 42.20% on AM (p<0.01), and by 56.06% on RM B-cells (p<0.01), which was significantly downregulated with VIP inhibition (both p<0.05). Similarly, CD95 expression on RM, TLM, and MN B-cells increased by 31.10% (p<0.001), 21.46% (p<0.01), and 39.92% (p<0.01) with R848 stimulation respectively, which was also significantly downregulated with VIP inhibition. Conclusion: These data indicate that B-cells in untreated HIV infection display increased levels of activation, and also the potential for increased susceptibility to apoptosis as evidenced by increased FAS (CD95) expression. VIP significantly down-regulated markers of activation, inhibition, and apoptosis signaling. Dysregulation of B-cells is thus apparent in asymptomatic stable chronic HIV-1 infection, which may impact on both inefficient neutralizing antibody production and hypergammaglobulinemia. The ability of VIP to prevent stimulationassociated marker upregulation may indicate that VIP is a potential therapeutic agent. Its immuno-modulatory properties were demonstrated to limit B-cell hyperactivation, and selectively down-regulate apoptosis and mark it out for further investigation. / AFRIKAANSE OPSOMMING: Inleiding: Immunaktivering en ongekoppelde immuun-homeostase is kenmerke van chroniese MIVinfeksie. Ons het perifere bloed B-sel subgroep-verspreiding, en veranderinge in die uitdrukking van merkers van aktivering, inhibisie, en apoptose in 'n onbehandelde MIV-1 besmettende groep ondersoek (in vergelyk met 'n gesonde onbesmettende kontrole). Die immuun-moduleerder, vasoaktiewe intestinale peptied (VIP) is bekend om aktivasie van geaktiveerde CD4+ T-selle te verminder, maar tot ons kennis, is daar geen studies wat die effek van VIP-inhibisie op B-sel aktivering ondersoek het, in die konteks van MIV-1 infeksie. Materiaal & Metodes: MIV+we individue (CD4-telling >250 selle/µl) , en MIVwe kontroles is gewerf uit die vrywillige toetsing en berading Emavundleni kliniek, Crossroads, Westelike Provinsie, Suid-Afrika. Bsel subgroepe is gedefinieer as geaktiveerde geheue (AM: CD21- CD27+ ), rusende geheue (RM: CD21+ CD27+ ), volwasse naïef (MN: CD21+ CD27- ), of weefsel-agtige geheue (TLM: CD21loCD27lo). Merkers van aktivering (CD126, CD86, CD38, CD284, CD287), inhibisie (CD72, CD85j, CD300a, CD305, CD307d), en apoptose signalering (CD95) is via vloeisitometrie (BD FACSCanto II) op B-selle ex vivo en ook op geïsoleerde B-selle (RosetteSep, Cell Technologies) ondersoek. Vir die bepaling van funksionele responsiwiteit, geïsoleerde B-selle (RosetteSep, StemCell Technologies) was vir 18h (37°C, 5%CO2) gekweek, sonder stimulasie of gestimuleer met TLR ligande (LPS of R848). Stimulasie eksperimente het ook in die teenwoordigheid of afwesigheid van VIP plaasgevind. Resultate: Chroniese MIV-1 infeksie het B-sel subset verspreiding geraak. Die persentasie (%) van TLM is verhoog deur 59,24%, en% RM het met 22.73% afgeneem (beide p <0,01). Totale uitdrukking van die VIP reseptor VPAC2 het met 47,35% afgeneem (p = 0,0296). Subgroepe het 'n gemengde ex vivo fenotipe; MIV-infeksie het CD38 (deur 59,56%, p=0,0004), CD72 (deur 60,70%, p=0,0396), CD307d (deur 68,63%, p=0,0015) op AM verhoog, terwyl RM Bselle het verhoogde uitdrukking van TLR4 (deur 107,04%, p=0,0057) en TLR7 (deur 208,14%, p=0,0199). TLM B-selle (die uitgeputtende fenotiep) het verhoogde TLR7 (deur 550%, p=0,0128) en CD307d (deur 72,40% p=0.045) uitdrukking gewys. MN B-selle het verhoogde uitdrukking van CD72 (deur 70,98%, p = 0,0026). R848 het CD86 uitdrukking op AM deur 42,20%, en op RM deur 56,06% toegeneem (beide p <0,01). Dit het met VIP inhibisie beduidend afgeneem (beide p <0.05). CD95 uitdrukking was soortgelyk verhoog op RM, TLM, en MN B-selle met 31.10% (p <0.001), 21,46% (p <0,01), en 39,92% (p <0,01) met R848 stimulasie. Al drie het beduidend afgeneem met VIP inhibisie. Gevolgtrekking: Hierdie data dui daarop dat B-selle in onbehandelde MIV-infeksie vertoon verhoogde aktiveringsvlakke, en ook die potensiaal vir verhoogde vatbaarheid vir apoptose soos bewys deur verhoogde uitdrukking van FAS (CD95). VIP het beduidend merkers van aktivering, inhibisie, en apoptose af-gereguleer. Wanfunksie van B-selle is dus in asimptomatiese stabiele chroniese MIV-1 infeksie duidelik, wat impak kan hê op beide oneffektiewe neutraliserende teenliggaampie produksie, en hiepergammaglobulinimie. Die vermoë van VIP stimulasie-verwante merker opregulasie te voorkom kan aandui dat VIP 'n potensiële terapeutiese agent is. VIP se immuno-moduleerende eiendomme is gedemonstreer om Bsel hieperaktiveering te beperk, en selektief apoptose afreguleer, en merk dit vir verdere ondersoek.

Tryptases are trypsin-like enzymes found in mast cell granules. Although in vivo substrates have not been positively identified, tryptases cleave a limited number of potential physiological substrates in vitro, including high molecular weight kininogen (HMWK) and vasoactive intestinal peptide (VIP). Purified human lung mast cell tryptase (HLT) apparently exists as a tetramer with an M$\sb{\rm r}$ of 135-144 kDa by gel filtration, whereas SDS-PAGE yielded two bands of M$\sb{\rm r}$ 29 Kda and 33 Kda. Tryptases are resistant to inhibition by most natural trypsin inhibitors and display some affinity for heparin. The existence of tryptase isozymes has been implied from the cloning of two tryptase cDNAs from human lung tissue, but distinct isozymes have not been isolated and characterized. This knowledge gap has been filled by isolating and characterizing two electrophoretically different forms of human lung mast cell tryptase, designated high-HLT (high molecular weight HLT) and low-HLT (low molecular weight HLT). These two forms of HLT have been separated by chromatography on a cellulose phosphate column, with the high M$\sb{\rm r}$ form eluting with 10 $\mu$M heparin and the low M$\sb{\rm r}$ form subsequently eluting with 1 M NaCl. Using HMWK and VIP as substrates, these two forms of HLT were found to differ with regard to specificity and rate of cleavage. High-HLT initially cleaved HMWK at a single Arg residue, whereas low-HLT cleaved HMWK simultaneously at multiple sites. Both isozymes cleaved VIP at multiple sites, but differed with regard to the preferential site of cleavage. Low-HLT was, on an active site basis, 25 and 2 times more active than high-HLT on HMWK and VIP, respectively. In addition, gel filtration of the isozymes yielded M$\sb{\rm r}$s of 125 Kda for high-HLT and 28 kDa for low-HLT, indicating tetrameric and monomeric quaternary structures, respectively. Both isozymes were inhibited by human secretory leukocyte proteinase inhibitor (SLPI), but not by other trypsin inhibitors tested. This work provides the first evidence for the existence of distinct tryptase isozymes, with supposedly different in vivo functions, and identification of an inhibitor that may control tryptase activity in vivo.

No description available.

Secretin and VIP stimulate cardiac adenylyl cyclase activity and exert a positive inotropic action in several mammalian species. This study examined positive inotropic activity and cAMP levels in rat papillary muscle. Isoproterenol and secretin increased contractions by 150 ± 31% and 129 ± 27%, respectively. VIP increased contraction by 30 ± 21% only at 10 μM. Isoproterenol significantly increased cAMP levels by 82%, whereas increases by secretin (58%) and VIP (56%) were not significant. These results are consistent with reports that secretin and VIP stimulate cardiac adenylyl cyclase in the rat, but suggest that cAMP tissue levels cannot totally explain the positive inotropic responses to secretin and VIP.

The molecular weight of the vasoactive intestinal peptide (VIP) receptor was assessed in bovine aorta, and rat liver, lung, and brain by covalent cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor in all four tissues was found to be a single polypeptide of approximate Mr 54,000, contradicting previous claims for substantial heterogeneity in the molecular weight of this receptor. Guanine nucleotides inhibit cross-linking of 125I-VIP to its receptor, and cross-linking with ethylene glycolbis(succinimidylsuccinate) provides further evidence for complex formation between VIP, its receptor and a guanine nucleotide-binding regulatory protein (G-protein). The precise mechanism of receptor-G-protein coupling may differ between the aorta and other tissues.

The parmacological effects of guinea pig vasoactive intestinal peptide (VIP) were studied in isolated perfused guinea pig hearts. Bolus injections of VIP produced a dose-dependent tachycardia that was not affected by atenolol. A decrease in amplitude of ventricular contractions occurred in response to all doses of VIP. This response was preceded by a small increase in amplitude in 3 of 6 hearts at the highest dose. VIP produced a decrease in perfusion pressure which was prominent after coronary tone was elevated with [Arg8]-vasopressin. The present findings support speculation that VIP may have a role in the regulation of heart rate and coronary blood flow.

The organization and transcription of the gene for vasoactive intestinal polypeptide (VIP) in rats and mice was investigated using northern- and Southern-blot hybridizations, selective genomic cloning, Sl-nuclease protection assays, oligonucleotide-directed RNase H digestions, and genomic cloning by standard methods. The center of the rat VIP gene and the entire mouse gene were cloned and sequenced. Selective genomic cloning was used to isolate a strongly-hybridizing fragment of the rat VIP gene identified in Southern-blot hybridizations with an existing human VIP cDNA. This fragment contains separate exons encoding VIP and a closely related neuropeptide, peptide histidine-isoleucine (PHI-27). This organization is the same in the mouse gene, which bears a total of seven exons and a close similarity to the human gene for VIP. Although the arrangement of exons suggested that VIP transcripts could be subjected to differential splicing to alter the coding capacity of the final messages, no evidence was found for this possibility. Two bands were seen in northern-blot hybridizations, but exon-specific probes and Sl-protection experiments provided evidence that they differed not in their coding regions but in the extent of their 3'-untranslated ends. RNase H digestions targeted to specific portions of transcripts from the VIP gene were used to resolve the principal band, to demonstrate that it represented a single species of message with sequence from both the VIP and PHI exons. In the course of the characterization of the murine VIP gene, a new method was developed for generating subclones for DNA sequencing in M13 bacteriophage. The central feature of the partial deletion subcloning method is its employment of frequent-cutting restriction endonucleases to detach different extents of the insert from a construction. The viral construct is first linearized at a unique site between the insert and the site for hybridization of the M13 sequencing primer. The linearized construct is then subjected to partial digestion with different frequent-cutting restriction enzymes. Partially digested products are repaired and religated. Products with deletions from the insert now have the sequencing priming site religated to a portion of the insert that formerly had been distant. Most of the products with deletions in the viral genome are not viable and do not survive the procedure. Subclones are sorted from the pool of transfected products by sizing of single-stranded viral DNA by agarose gel electrophoresis. Selected subclones are subjected to a simple test for the presence of the sequencing priming site. With this method and its associated tests, a variety of restriction enzymes can be used to generate a spectrum of deletion subclones for sequencing. In a simple trial of this method with an unknown 3.3 kilobasepair cDNA, a set of subclones was generated to allow sequencing to span the cDNA in one direction.

Although inhibitory cortical interneurons play a critical role in regulating brain excitability and function, the effects of stroke on these neurons is poorly understood. In particular, interneurons expressing vasoactive intestinal peptide (VIP) specialize in inhibiting other classes of inhibitory neurons, and thus serve to modulate cortical sensory processing. To understand how stroke affects this circuit, we imaged VIP neuron responses (using GCaMP6s) to low and high intensity forepaw stimulation, both before and after focal stroke in somatosensory cortex. Our data show that the fraction of forelimb responsive VIP interneurons and their response fidelity (defined as a cell’s number of responsive trials out of eight trials at a certain imaging week) was significantly reduced in the first week after stroke, especially when lower intensity forepaw stimulation was employed. The loss of responsiveness was most evident in highly active VIP neurons (defined by their level of responsiveness before stroke), whereas less active neurons were minimally affected. Of note, a small fraction of VIP neurons that were minimally active before stroke, became responsive afterwards suggesting that stroke may unmask sensory responses in some neurons. Although VIP responses to forepaw stimulation generally improved from 2-5 weeks recovery, the variance in response fidelity after stroke was comparatively high and therefore less predictable than that observed before stroke. Lastly, stroke related changes in response properties were restricted to within 400μm of the infarct border. These findings reveal the dynamic and resilient nature of VIP neurons and suggest that a sub-population of these cells are more apt to lose sensory responsiveness during the initial phase of stroke, whereas some minimally responsive cells are progressively recruited into the forelimb sensory circuit. Furthermore, stroke appears to disrupt the predictability of sensory-evoked responses in these cortical interneurons which could have important consequences for sensory perception. / Graduate / 2021-01-13

Angiogenesis is thought to play a pivotal role in the cycling endometrium. Coordinated by oestrogen and progesterone, endometrial blood vessel development is primarily mediated by vascular endothelial growth factor-A (VEGF-A), which promotes endothelial cell (EC) proliferation and protects ECs from induced apoptosis. Studying changes at transcript level in human endometrial endothelial cells (HEECs) in response to mitogenic and inhibitory stimuli is one way towards understanding the regulation of physiological endometrial angiogenesis. Endometriosis, the presence of endometrial-like tissue outside the uterine cavity, is a common gynaecological disorder in women of reproductive age, often causing pelvic pain and reduced fertility. Chronic inflammation in the peritoneal environment and defective endometrial protein expression are some of the contributors to the complex pathophysiology of endometriosis. The aim of this work was to study the changes in the transcriptome induced by VEGF-A and partial serum deprivation in primary HEECs, and to investigate biochemical factors associated with subfertility and chronic pelvic pain in endometriosis patients. Exposing primary HEECs to VEGF-A, and serum withdrawal was found to regulate transcripts associated with survival, migration, apoptosis and progression through the cell cycle, when assessed using microarray technology and bioinformatic tools. A subset of 88 transcripts was reciprocally regulated under the two experimental conditions; thus probably important in HEEC biology. Higher endometrial epithelial staining scores of oestrogen receptor-α and reduced staining of progesterone receptors were seen in subfertile endometriosis patients. Lower levels of the receptivity biomarker leukaemia inhibitory factor (LIF) and its receptor, as well as signs of dysregulated αB-crystallin expression and increased peritoneal fluid concentrations of interleukin (IL)-1α and IL-6 were associated with reduced pregnancy rates. Endometriosis patients with chronic pelvic pain had higher levels of vasoactive intestinal peptide (VIP) in eutopic endometria and in endometriotic lesions compared with patients without chronic pain. The presence of chronic pelvic pain was also associated with increased concentrations of VIP and IL-6 in peritoneal fluid. The present results may constitute a basis for further investigation of regulatory pathways in endometrial angiogenesis as well as for studies of endometrial receptivity and pain in women with endometriosis.