Regular ovarian and cancer stem cells Functional assays Isolation of SC in the theca and ovarian surface epithelium is feasible lately. Thecal stem cells have been obtained just after dissociating newborn mice ovaries and developing them in serum absolutely free germline stem cell media. Nonadherent anchorage independent spheres exhibited ideal gene profiles, compatible with theca cells that differentiate into early precursors and steroidogenic cells inside a stepwise method right after treatment method with serum, luteinizing hormone, and paracrine elements from granulosa cells, and later secreted androstenedione. At each step these cells displayed appropriate gene expres sion profiles and morphological functions and achieved a mature morphology when coculture with isolated granulosa cells. Also, they colonized exclusively the ovarian interstitium along with the theca layer of follicles when transplanted into ovaries of recipient animals.
A population of label retaining cells residing from the coelomic selleck epithelium and exhibiting quiescence, in vivo practical response to hormonal stimulus, and enhanced in vitro colony formation are actually identified as candidate for somatic stem progenitor cells from the mouse ovary. Existence of ovarian CSCs is supported by identifica tion and isolation of tumorigenic sphere forming clones from ascites of sufferers with epithelial ovarian cancer. Immunohistological proof advised differenti ation along epithelial, granulosa, and germ cell lineages. Independent clones showed an ability to kind spheroids and multicellular colonies in soft agar correlating with tumorigenicity. Xenografted tumors may be serially passaged as a result of at the very least 3 generations in vivo, indicating their capability to self renew.
Markers Ovarian CSCs were noticed to type tumors more quickly and with much less inoculums, when injected in to the dorsal excess fat pad of nude mice. M?llerian inhibiting kinase inhibitor KU-0060648 substance was able to reduce the growth of those cells in vitro. Surface proteins including c Kit, CD44 and CD133 have been related with ovarian cancer cells with stem like phenotype. Expression of CD133 1 and CD133 2, which were detected in ovarian carcinomas, was also observed in usual ovaries. CD133 ovarian tumor cells had been characterized by a increased proliferative likely and clonogenic efficiency than damaging cells. CD133 cells from cancer cell lines, main tumors and ascitic fluid of ovarian cancer sufferers had been shown for being tumorigenic. CD133 cells derived from ovarian tumors have been capable of self renewal and had been related with elevated tumor aggression in xenografts. On top of that, they recognized that epigenetic deregulation of CD133 may perhaps be associated with transformation.

ADAMTS four and ADAMTS 5 protein down regulation at 56 days was even further inconsistent with their elevated or constant mRNA expression. These ADAMTS findings probably correlate with decreased aggrecanase cleaved aggrecan neoepitope. The observed discrepancy in between mRNA and protein expression during the NP might be explained by prolonged, prominent MMP induced aggrecanolysis and shortened, modest aggrecanase induced aggrecanolysis. on the other hand, even more investigations are required to understand the regulation mechanism of MMPs, ADAMTSs, and TIMPs on the post transcriptional level. The pathomechanism of AF degradation has not been clarified in detail. The annulus matrix comprises col lagens, proteoglycans, and elastic fibers elastin and microfibril for example glycoprotein fibrillins. Elastin is readily degraded by MMP two, MMP 3, MMP seven, MMP 9, MMP 10, and MMP twelve. Fibrillins are degraded by MMP 2, MMP 3, MMP 9, MMP 12, MMP 13, and MMP 14.
This study lacked actual time RT PCR ana lysis of AF tissue, which can be an inherent discover more here limitation. In immunohistochemistry, the prominent MMP 3 TIMP one and TIMP two imbalance having a per sistent boost of MMP cleaved aggrecan neoepitope was observed inside the AF too as in the NP. Meanwhile, the ADAMTS four and ADAMTS 5 TIMP three imbalance in the AF had a pattern various from that while in the NP. extra impressive ADAMTS 4 and ADAMTS 5 up regulation than TIMP 3 down regula tion was detected. This imbalance appeared to produce a transient improve of aggrecanase cleaved aggrecan neoepitope at very much the same time as the NP. The histomorphological research by Boos and colleagues showed the NP was more severely degenerated during the exact same age group than the AF. nevertheless, our biological findings indicate AF degen eration occurs simultaneously with NP degeneration.
Our rat tail immunohistochemical results are summar ized in Figure six. Each imbalances of MMP 3 TIMP 1 and TIMP 2 and ADAMTS 4 and ADAMTS 5 TIMP 3 operate within the early to middle stages of seven and 28 days. Nefiracetam yet, the MMP 3 TIMP one and TIMP two imbalance is a lot more significant than the ADAMTS four and ADAMTS 5 TIMP 3 imbalance at the late stage of 56 days. This pro vides a sound argument for low aggrecanase action within the discs with innovative degeneration. In ECM metabo lism, the relative significance of MMPs and aggrecanases has extended been debated. Minor and colleagues reported catabolic aggrecan degradation in usual and osteoar thritis cartilage generally involved cleavage by aggreca nase and not by MMPs. It had been observed by Karsdal and colleagues that MMP mediated degradation of aggrecan and collagen style 2 brought about irreversible harm for cartilage, though aggrecanase mediated degradation of aggrecan was completely reversible. Integrated with these reviews, our findings demonstrate that a state of dominant MMP 3 TIMP one and TIMP two imbalance relative to ADAMTS 4 and ADAMTS 5 TIMP 3 imbalance may possibly indicate an irreversible stage of intervertebral disc degeneration.

Hofer et al. identified no correlation concerning baseline sVCAM 1 and mortality in septic sufferers but reported considerably larger sVCAM 1 amounts at 48 and 120 hrs in non sur vivors when compared with survivors. Only one review addressed correlation of sVCAM 1 with clinical severity scores, and reported modest corre lation with SOFA and APACHE II. Two scientific studies reported variability of sVCAM 1 in sepsis across various patient populations. Presterl et al. investigated the difference of sVCAM 1 degree in Can dida sepsis compared to bacterial sepsis, and discovered that sVCAM 1 was greater in Candida sepsis at days one, seven and 14. Similar to sICAM 1, Endo et al. noticed higher sVCAM 1 ranges with escalating age, and observed the dynamics of serial sVCAM one have been numerous in sufferers stratified by age. Especially, sVCAM one values greater more than the program of sepsis time in older sufferers and decreased in younger patients.
One particular review uncovered that sVCAM 1 was not linked with left ventricular size or function in patients with sepsis or septic shock. Soluble E selectin Twenty 3 studies were recognized that evaluated selleck Lenalidomide sE selectin as a biomarker in sepsis. Association with sepsis The majority of recognized scientific studies reported larger amounts of sE selectin in sepsis in comparison to healthful controls or other patient groups devoid of sepsis. Ten research specifi cally reported significantly elevated sE selectin ranges in sepsis when in contrast with wholesome controls. Geppert et al. reported increased sE selectin amounts in sufferers with SIRS following cardiopulmonary resuscitation compared to controls. sE selectin was also reported to be substantially higher in septic individuals in comparison with trauma individuals, ICU controls, sufferers with infection but without systemic sepsis, patients with shock from other leads to, and individuals with many organ failure without infection.
Hynninen et al. concluded that sE selectin values have been not statistically distinct in individuals with serious sepsis from these with extreme acute pancreatitis. Association with clinical selleck final result The reported association of sE selectin and disorder severity continues to be inconsistent. Five studies showed a correlation in between the marker and escalating sepsis severity, although 3 studies didn’t find a substantial correlation. Thirteen of the identified studies evaluated the asso ciation between sE selectin and mortality, with 9 stu dies reporting a significant beneficial correlation and four research reporting no correlation. Among the research reporting good association, there was substantial heterogeneity inside the power and kind of association. 1 study of ICU sufferers with serious sepsis and septic shock reported that baseline sE selectin one amounts were increased in non survivors than survivors, but the variation existed only for the 1st three days of sepsis.

Differences have been thought to be considerable in the event the P value was 0. 05. For that intravasation assay, the Mann Whitney Wilcoxon rank sum check was utilized in addition on the Pupil t check. Final results Gene expression profile of migratory human tumor additional resources cells in vivo the human invasion signature We previously showed that we are able to collect the migratory cells from MDA MB 231 principal tumors in response to epidermal growth issue or colony stimulating issue one through the use of an in vivo invasion assay. In brief, microneedles containing a chemoattractant are positioned in main tumors while the tumor bearing mouse is alive and underneath anesthesia. This generates a che motactic gradient similar to physiological stimuli within the main tumor, shown to initiate tumor cell invasion. Indeed, we previously reported that chemotaxis and lively migration are demanded to the tumor cells to enter the microneedles.
Hence, this assay exams the cells capability in vivo to undergo chemotaxis towards a chemo kine gradient, to invade with the tumor matrix, and ultimately to migrate in excess of extended distances towards the source of the gradient. For brevity, the Carfilzomib tumor cells collected with this particular assay are going to be hereafter identified as migratory tumor cells. With this assay, we recently showed that the inva sive properties within the MDA MB 231 human breast ade nocarcinoma cells differ in vitro and in vivo, due to the fact of a TGF b initiated autocrine CSF1 CSF1R loop that happens from the tumor microenvironment. We also showed that this hypermotile tumor cell subpopulation sponta neously expresses an invasion exact isoform of Mena, that is the hallmark of migratory tumor cells in mammary tumors. This emphasizes the importance of isolating the migratory tumor cells immediately through the principal tumor in vivo, to understand their complete probable and characteristics.
With this particular in vivo invasion assay, we isolated the migra tory tumor cells from orthotopic MDA MB 231 tumors after which compared their gene expression profile by microarray examination with the total or average primary tumor cell population, and that is principally nonmigratory. Total, 443 transcripts had been uncovered for being significantly altered in the migratory tumor cells, of which 185 were annotated genes with known protein items. We define this gene record because the human invasion signature. To gain insight to the biologic properties of the migratory breast tumor cells, Ingenuity Pathway Evaluation was implemented first to rank enriched functional classes of gene networks relating to your transcripts regulated within the HIS. Table one displays the leading 5 most sig nificantly upregulated and downregulated functions relevant to the gene networks of your HIS, along with the record within the corresponding genes in each and every perform network.

We chose these genes because they encode functions which might be known to be, or potentially associated with, regulatory pathways that control re epithelialization, innate immunity, cell form, and cell tension responses. Employing in situ hybridization, these candidate genes had been examined for wound dependent epidermal transcriptional activation at a single hour after puncture wounding of stage 15 17 wild sort embryos. Eight of these 27 candidate genes had been transcriptionally upregulated in epidermal cells surrounding the wound web-site. Thus, trypsin mediated wounding allowed us to determine the next 8 further localized epidermal wound response genes Ady43A, Ets at 21C, jun relevant antigen, kayak, Relish, rhomboid, spatzle, and dorsal. A lot of these genes wouldn’t have already been selected as candidates with out the trypsin amplification outcomes, as puncture only microarray expression levels were not statistically substantial at most time points for 5 of the eight genes tested.
Ady43A transcripts are undetectable above background in unwounded embryos, but following puncture wounding transcripts accumulate inside a broad zone of epidermal cells around wound web-sites. INK1197 ic50 Ets21C transcripts are observed ubiquitously at low amounts from the epidermis and inside the ventral nerve cord in wild type embryos. soon after puncture wounding Ets21C transcripts are detected at greater levels inside a narrow zone of epidermal cells all around wound web sites. Jra jun transcripts are detected at reduced levels in all cells of unwounded embryos, but after puncture wounding jra jun transcripts are detected within a narrow zone of epidermal selelck kinase inhibitor cells all over wound online websites. In unwounded embryos, kay fos transcripts are detected at large amounts while in the late embryonic midgut hindgut, and at very low levels through the entire epidermis.
following puncture wounding kay fos transcripts are activated inside a moderately broad zone of epidermal cells around wound internet sites. Spz RNA is expressed inside the producing rectum of unwounded embryos. soon after puncture wounding, spz transcripts may also be detected in the broad zone of epidermal cells all over wound sites. Transcripts from dl are expressed weakly throughout the head and thoracic epidermis along with other anterior tissues of unwounded embryos. immediately after puncture wounding, dl transcripts are considerably upregulated in a moderately broad zone of epidermal cells close to wound sites. Rho RNA is detected from the peripheral nervous strategy of late stage unwounded embryos. soon after puncture wounding, rho transcripts are upregulated within a narrow zone of epidermal cells around wound websites. Rel RNA is expressed at low ranges while in the midgut and excess fat physique of unwounded embryos.

Statistical Evaluation Statistical analyses for gene expression studies are described above. For cytokine measurements, paired information have been evaluated using a 2 tailed Students t test. Differences having a P worth lower than 0. 05 had been deemed statistically sizeable. Effects Primary Human B Cells Contribute for the Polarization of CD4 T Cells to a Th17 Phenotype in a Model of T Cell Dependent B Cell Activation We have previously described a co culture assay with primary human B cells and PBMC stimulated having a IgM along with a reasonably low concentration of the SEB and TSST 1 super antigens that designs T cell dependent B cell activation. The reduced concentration of SAg utilized in this model facilitates T cell dependent B cell activation with minimal results on T cell proliferation. This concentration of SAg will allow us to interrogate the mechanisms that regulate T cell cytokine production independently of T cell proliferation depen dent effects.
SAg also masks any allogeneic reaction that could arise from mixing cells from various donors. In characterizing this model, we measured genome broad mRNA expression amounts by microarray in B cell and PBMC co cultures soon after three days of stimulation by using a IgM selleck and SAg. Interestingly, Il17f was probably the most strongly induced gene in co cultures right after three days of stimulation. This acquiring suggests that activation ailments appropriate for T cell dependent B cell activation also contribute to B cell dependent T cell responses, leading to the production of IL 17 loved ones cytokines by 1 or even more cell sorts. To find out which cell types in BT co cultures had been generating IL 17 relatives cytokines, we performed intracellular movement cytometry for IL 17A and IL 17F with cell surface markers unique for CD4 T, CD8 T, B, NK, and NKT cells.
Detection of IL 17A and IL 17F by intracellular movement cytometry needs secondary stimulation with phorbol twelve myristate 13 acetate and ionomycin in combination having a protein transport inhibitor, this kind of as monensin. Even so, a limitation of this system is the fact that secondary stimulation triggers decreased surface expression of CD4, which undermines CCI-779 the detection of CD4 T cells. We therefore applied the gating tactic proven in Figure 1, whereby CD4 T cells are detected just after 1st gating over the total CD3 cell population and then analyzing the cells that are unfavorable for CD8 staining. Virtually every one of the cells labeled with antibodies to IL 17A and IL 17F expression. IL 17A or IL 17F antibodies had been of the mouse IgG1, k isotype along with a mouse IgG1, k isotype handle antibody utilized in place of antibodies to IL 17A or IL 17F exhibited a minimal intracellular cytokine signal. These information indicate that CD4 T cells are the predominant cell sort that creates IL 17A and IL 17F in this model of T cell dependent B cell responses.

In PAH model immediately after chronic hypoxia exposure, the pathology on the pulmonary vasculature was grossly typical as reported, which includes, e. g. medial thickening and muscularization of modest arteries inside the alveolar walls, along with the grow of collagen fibers both in WT and HEX Tg mice. The extent of elevation in RV systolic stress, plasma concentrations and mRNA expression inside the lungs of ET one, were also related. We, consequently, concluded that PAH was similarly produced in both mice. Concerning cardiac phenotype, on the other hand, the degree of RVH was significantly less marked in HEX Tg mice in contrast with WT mice. the RV excess weight to left ventricular and septum weight ratio and RV weight to body weight ratio weren’t appreciably greater in HEX Tg mice. LV S bodyweight to BW ratio BW was comparable between WT and HEX Tg mice.
In WT mice, the diameter of cardiomyocytes in RV wall have been significantly increased under exposure to persistent hypoxia, supporting the previous notion that afterload driven RVH is due to not elevated within the number of myocytes but for the selleckchem elevated cell size. In clear contrast, HEX Tg mice did not present substantial enlargement of myofiber diameter. We evaluated RV function by cardiac ultrasonography and unveiled sizeable RV dilatation not in HEX Tg mice but solely in WT mice. Under this affliction, left ventricular ejection fraction was not impaired in both mouse. Taken together, these findings indicated that cardiomyocyte unique overexpression of HEXIM1 inhibits progression to RVH under continual hypoxia, most quite possibly by way of inhibition of P TEFb mediated enlargement of cardiomyocytes. Discussion Within the fetus, cardiovascular physiology is characterized by a high resistance pulmonary circulation and low resistance systemic circulation.
Right after birth and in infancy, RVH regresses as well as the heart remodels to your typical postnatal heart that has a crescent shaped RV and elliptic LV. Interestingly, selelck kinase inhibitor HEXIM1 is extremely expressed from the fetus and early postnatal time period and its expression is gradually decreased. Thinking about that HEXIM1 could possibly possess adverse result on cardiomyocyte growth, it is very likely that this developmental stage dependent alteration in HEXIM1 expression amounts might be connected with physiological cardiovascular development. On top of that, we showed that PGI2, a therapeutic drug for PAH, increases HEXIM1 amounts in cardiomyocytes. Given that PGI2 is identified to negatively modulate RV remodeling in experimental PAH animals and PAH individuals, we hypothesized that HEXIM1, probably through suppression of P TEFb, takes part in cardiomyocyte regulation in RV. Despite a number of reports with loss of function experiments, it remains unclear no matter whether boost of HEXIM1 expression amounts, as a physiological inhibitor of P TEFb, can exert antihypertrophic result in cardiomyocytes.

With this particular representation is possible to have an overview on genes which display either equivalent or differential expression trends within the two species. A listing of the genes exhibiting by far the most substantial differences is obtainable in Table S5. The Notch pathway. The Notch pathway is really a very conserved cell signaling process current in most multicellular organisms. it influences differentiation, proliferation, apoptotic events in any way phases of improvement, and importantly it’s also been implicated in numerous elements of stem cell biology. Notch controls the cell fate possibilities according to the differential expression of ligands and receptors in opposing cells. We analyzed 27 genes existing within the pathway Advancement Notch Signaling Pathway in GeneGo. Within the comparison B vs M a lot of the 27 genes behaved comparable in each species.
Our research having said that identifies Notch1, 1 with the 4 acknowledged Notch receptors, remaining upregulated within the mouse and downregulated while in the rat from the comparison ICM vs B at the same time as in ICM vs M. So, Notch1 is upregulated inside the mouse ICM but downregulated within the rat ICM. Activated Notch1 can encourage, dependent in the context, both differentiation processes or servicing of stem cell proliferation. Therefore, thanks to its range of molecular pan JAK inhibitor functions, the obtaining that Notch1 is differentially expressed inside the mouse and rat preimplantation embryos, specifically while in the cells from the ICM, suggests a achievable diverse purpose of this gene within the two species. We also observed considerable alterations inside the expression ranges of other 5 genes Aph1a, Jag1, Maml1, Tle2, and Tle4. The gene Aph1a was upregulated from the mouse but only slightly transformed during the rat while in the comparison B vs M. This gene encodes the membrane protein APH 1 that is definitely an vital member in the c secretase, which cleaves single pass transmembrane proteins at residues inside the transmembrane domain.
APH 1a is definitely the key mammalian APH one isoform needed for Notch signaling throughout embryogenesis. Interestingly, the Notch ligand Jag1 was downregulated through the transition from morula to blastocyst stage in the two species, but this downregulation was stronger from the EX-527 rat embryos than in the mouse. The gene Maml1 is concerned within the regulation on the transcriptional activation of Notch target gene expression. Maml1 was strongly upregulated from the mouse while in the comparison B vs M and ICM vs M indicating that Maml1 expression increases in the morula towards the blastocyst stage. From the rat on the other hand, Maml1 was specifically upregulated from the cells within the ICM highlighting once more a potentially diverse activation with the Notch pathway during the two species. The most essential targets with the Notch1 complex will be the HES genes, which are transcriptional repressors that depend upon the standard corepressor Groucho Transducin like enhancer of split protein family members.

Additionally, microbial get in touch with or invasion could be confined to elements from the alimentary tract which are reasonably inaccessible to tissue sampling, this kind of as the ileum or jejunum. The in vivo perinatal calf jejunal ileal loop model supplies an ideal animal model to even more precisely review the early pathogenesis of MAP or other microbial pathogens in Crohns sickness. Inside the present research, we hypothesized that MAP induces an immune tolerance in Peyers patch very soon immediately after invasion. To test our hypothesis, we analyzed the temporal gene expression in detail throughout the early stages of colonization of Peyers patch by MAP. We utilized a techniques biology approach to analyze the complex microarray information enabling us to recognize precise cellular pathway perturbations and predicted cell style involvement during the early infection and colonization.
In addition, we recognized innate a cool way to improve response signatures satisfactory to envisage the subsequent adaptive immune responses resulting in persistent MAP infection. The aims of this research were two fold, one to employ a methods biology analytical technique to achieve new insights regarding just about the most perturbed cellular pathways. and two to supply proof for tolerance relevant parts of mucosal immunity throughout early MAP infection. Final results Invasion of Ileal Mucosa by MAP MAP was recovered from your MAP inoculated ileal tissues at all the time points post infection. No bacteria had been detected from the PBS inoculated loops. Between the contaminated loops, no significant modifications within the amount of MAP had been detected at any occasions post inoculation. Bovine Peyers Patches Inoculated with MAP Reveals a Complicated Temporal Pattern of Transcriptional Profile So as to attain thorough insight in to the alterations within the transcriptional profile of genes in bovine intestinal Peyers patch mucosa inoculated with 36109 cfu of MAP, initially the microarray information examination was performed through the use of GeneSifter software.
Classical examination from the altered gene expression by GeneSifter, supplies the static changes while in the experimental ailments by using a profound spectrum of information. on the other hand, after filtering the information to the biological OSI-930 c-Kit inhibitor relevant and significant genes, a limited variety of genes have been found to have statistically substantial expression. As proven in Table one, no genes were significantly up regulated in MAP contaminated animals with the earliest time point tested, and only modest numbers of genes were greater in expression in excess of the experimental time program. In contrast, on the earliest time factors, MAP contaminated loops had down regulation in gene expression. This observation plainly reflected that measurement of subtle improvements may be undetected if one the amount within the adjust is incredibly smaller to measure, or 2 if experiments are carried out utilizing out bred populations, or 3 should the sample applied is a heterogeneous population of a few cell forms expressing numerous levels of expression of similar gene.

Considering that EGFR in excess of expression and amplification on the EGFR locus has become linked towards the presence of EGFRvIII, we utilised quantitative immunohistochemistry and AQUAH technologies to measure EGFR expression in TMAs constructed from our patient tumor samples. The single OSCC patient that tested positive for EGFRvIII also exhibited the highest expression of EGFR protein. This improved staining could possibly be attributed to your presence of each wild form and mutant varieties of EGFR, considering the fact that our EGFR antibody detects both wild sort EGFR and EGFRvIII. Also, the absence of EGFRvIII in an additional tumor sample through the similar patient, and expressing significantly reduce levels of EGFR, corroborates reviews suggesting that EGFR in excess of expression is linked for the presence of EGFRvIII.
The obvious mosaic pattern of EGFRvIII expression in the EGFRvIII good patient also confirms the heterogeneity of OSCC similar to malignant glioblastomas, the place the proportion of EGFRvIII selleck inhibitor expressing cells is proven to vary from 37% to 86% in EGFRvIII constructive tumor samples. The cancer certain EGFRvIII deletion mutant will be the concentrate of investigation in the number of cancers. In vitro and in vivo studies attest for the improved tumorogenicity of EGFRvIII expressing cells, as a result making it a prime target for novel therapies. Yet, the implementation of these therapies is contingent on the exact and delicate detection of EGFRvIII in tumor samples. Various tactics can be found for your detection of EGFRvIII in clinical specimens. Fresh frozen tissue samples are amenable to conven tional RT PCR and direct sequencing based mostly detection tactics but these approaches are unreliable in FFPE samples. RNA isolated from paraffin embedded tissue is usually very degraded and compromised by cross linking and modifications introduced throughout the fixation process.
This could show challenging for downstream applications this kind of as PCR exactly where higher nucleic acid integrity is vital. Immunohistochemical detection of EGFRvIII protein can realize large sensitivity and specificity, attributed for the presence of the one of a kind glycine residue in EGFRvIII. Nevertheless, the widespread clinical utilization of EGRFvIII immunohistochemistry GSK1059615 is restricted by patents and availability of antibodies. A number of real time PCR primarily based strategies have also been described from the literature and their utility in FFPE tumor samples has been successfully demonstrated. A majority of those strategies use dye based mostly chemistries and are much less specific than probe primarily based assays, rendering them a lot more prone to the detection of false positives. Also, the sensitivity of a few of these serious time PCR based mostly solutions may perhaps be inadequate for detecting trace quantities of EGFRvIII mRNA that may be existing in early stage tumor samples. Our novel rt RT PCT assay is less vulnerable to the detection of false positives on account of its specificity for EGFRvIII.