Thymus Ontogeny in Frogs: T-Cell Renewal at Metamorphosis

Metamorphosis in amphibians presents a unique problem for the developing immune system. Because tadpoles are free-living, they need an immune system to protect against potential pathogens. However, at metamorphosis, they acquire a variety of new adultspecific molecules to which the tadpole immune system must become tolerant. We hypothesized that Xenopus laevis tadpoles may avoid potentially destructive antiself responses by largely discarding the larval immune system at metamorphosis and acquiring a new one. By implanting triploid (3N) thymuses into diploid (2N) hosts, we examined the influx and expansion of host T-cell precursors in the donor thymus of normally metamorphosing and metamorphosis-inhibited frogs. We observed that donor thymocytes are replaced by host-derived cells during metamorphosis, but inhibition of metamorphosis does not prevent this exchange of cells. The implanted thymuses export T cells to the spleen. This donor-derived pool of cells declines after metamorphosis in normally developing frogs but is retained to a greater extent if metamorphosis is inhibited. These studies confirm previous observations of a metamorphosis-associated wave of expansion of T cells and demonstrate that it is not dependent on the relatively high concentrations of thyroid hormones required for metamorphosis. Although some larval T cells persist through metamorphosis, others may be destroyed or the larval population is significantly diluted by the expanding adult population.     

Developmental and Anatomical Patterns Of IL-2 Gene Expression in Vivo in The Murine Thymus

Interleukin-2 (IL-2) is a potent growth factor that mature T lymphocytes synthesize and use as a proliferation signal. Much controversy has arisen concerning whether it is used to drive the extensive proliferation of immature pre-T cells in the thymus. Immature thymocytes acquire the competence to express IL-2 at an early stage, but it has remained uncertain whether they are activated to exercise this competence in vivo. Therefore, we have used in situ hybridization and immunohistochemistry on serial sections obtained from fetal and adult thymuses of normal C57BL/6 mice and of mice bearing the scid defect to determine where, when, and whether IL-2 is expressed in vivo. Our results show a striking spatial and temporal pattern of IL-2 expression in the normal fetal thymus. We detected a burst of IL-2 mRNA accumulation at day 14.5 of gestation, which rapidly decreased by day 15. At day 15, we observed maximal IL-2 protein production that subsequently decreased by day 16 of gestation. Both in situ hybridization and immunohistochemical staining revealed an unexpectedly strict localization of IL-2 expressing cells to patches around the periphery of the fetal thymus, creating a previously unrecognized compartment of high IL-2 protein content. IL-2 production in the day-15 fetal thymus appeared to be unaffected by the scid mutation, indicating that this response is likely to be T-cell receptor (TcR)-independent. Several features distinguish the IL-2 induction pattern in the adult thymus from that in the fetal thymus. In the normal adult thymus, IL-2-expressing cells are extremely rare (found at a frequency of 10^-7), but they are reproducibly detectable as isolated cells in the outer cortex and subcapsular region of the thymus. Unlike the fetal thymic IL-2 producers, the IL-2 producers in the adult thymus are completely eliminated in mice homozygous for the scid mutation. This suggests that the IL-2-expressing cells in the normal adult thymus are of a more mature phenotype than the immature, TcR-negative cells that accumulate in the scid adult thymus. Thus, our work demonstrates that two developmentally distinct types of cell interactions induce IL-2 expression in vivo: one, a broadly localized interaction in day 14‑15 fetal thymus that is unaffected by the scid mutation; the other, a rare event that occurs asynchronously from late fetal through adult life, but which is completely eliminated by the scid defect. These results imply that significant differences exist between the physiological processing of thymocytes in the fetal and postnatal thymic microenvironments.     

Analysis by in Situ Hybridization of Cells Expressing mRNA for Tumor-Necrosis Factor in the Developing Thymus of Mice

We have used in situ hybridization to investigate the expression of TNF-α genes by thymic cells during fetal development in mice. In 14-day-old fetal thymuses, very scarce cells produce TNF-α mRNA. A second phase of cytokine gene expression starts on day 16. The density of positive cells progressively increases up to day 20. Thymuses at 15 days of gestation and after birth do not express detectable cytokine mRNA. In an attempt to identify the nature of the TNF-α mRNA-producing cells, acid phosphatase activity, which is characteristic of the macrophage lineage, was studied in the same thymuses. Acid phosphatase-positive cells only appear on day 15. Their frequency increases up to birth. However, no correlation can be established between acid phosphatase—and TNFα mRNA— positive cells. The results indicate that a small subset of thymic cells is responsible for TNF-α mRNA production during ontogeny: These cells are not yet identified. The possible role of TNF-α in thymic ontogeny is discussed.     

Expression of The αβ T-Cell Receptor Is Necessary for The Generation of The Thymic Medulla

The architecture of the thymus of mice that congenitally fail to express the αβ T-cell receptor (TCRαβ) has been examined by immunohistology. In these mice, a defined mutation was introduced into the TCRc gene by homologous recombination. By using antibodies specific for cortical or medullary epithelium and for major histocompatibility complex antigens, the network of cortical epithelium in these mice was shown to be essentially unaltered in comparison with that of normal mice. In contrast, the thymic medulla was considerably reduced in size. This analysis shows that expression of the αβ TCR but not the γδ TCR is obligatory for establishing the thymic medulla and suggests that the growth of medullary epithelial cells may require contact with TCRαβ-expressing cells.     

Multi-disciplinary treatment of a rare pelvic cavity ependymoma

Ependymomas usually develop from neuroectodermal organs. Here, we present an ependymoma arising from the pelvic cavity. A 27-year-old Korean female was admitted to the hospital with a sensation of abdominal fullness. Imaging studies revealed a huge heterogeneous nodular mass in the pelvis and lower abdomen. Laparotomy showed that two large masses with multiple nodules were located between the uterus and rectum and uterus and bladder, respectively. Histologically, the tumor was characterized by compact columnar neoplastic cells divided by fibrovascular septae. The neoplastic cells formed true ependymal rosettes and perivascular pseudorosettes. Immunohistochemical staining showed a strong positive reaction for glial fibrillary acidic protein (GFAP) and vimentin and a partial positive reaction for S100 and EMA. The tumor was thus diagnosed as an ependymoma arising from the pelvic cavity. The patient was treated with a debulking operation and chemotherapy based upon the in vitro chemosensitivity test results. The patient was free of cancer for 4 years following surgery. This is a rare case of extraneural ependymoma for which an in vitro chemosensitivity test was critical in determining the multidisciplinary approach for treatment.     

Prion propagation in vitro: are we there yet?

Prion diseases are caused by proteinaceous pathogens termed prions. Although the details of the mechanism of prion propagation are not fully understood, conformational conversion of cellular prion protein (PrP^C) to misfolded, disease-associated scrapie prion protein (PrP^Sc) is considered the essential biochemical event for prion replication. Currently, studying prion replication in vitro is difficult due to the lack of a system which fully recapitulates the in vivo phenomenon. Over the last 15 years, a number of in vitro systems supporting PrP^C conversion, PrP^Sc amplification, or amyloid fibril formation have been established. In this review, we describe the evolving methodology of in vitro prion propagation assays and discuss their ability in reflecting prion propagation in vivo.     

Expression, Genomic Structure and Mapping of the Thymus Specific Protease Prss16: A Candidate Gene for Insulin Dependent Diabetes Mellitus Susceptibility

PRSS16 is a serine protease specifically expressed by epithelial cells in the thymic cortex. The human gene is encoded on 6p21.3-p22 where recent linkage analysis has identified an association with insulin dependent diabetes mellitus (IDDM) susceptibility independent of HLA-DR3. To further investigate its potential role in autoimmunity, we characterized the mouse orthologue, Prss16. The genomic structure of Prss16 shows conservation with the human gene in size, number of exons and chromosomal location. Mapping of Prss16 places it on mouse chromosome 13 centromeric of thesatin locus. This region is comparable to the PRSS16 region on human chromosome 6 and has also been linked to quantitative trait locus for IDDM in the nonobese diabetic mouse. Similar to the human gene, Prss16 expression is highly specific in the mouse with expression limited to the cortical thymic epithelium. Notably, embryonic expression coincides with population of the thymic anlage with T-cell precursors and initiation of T-cell development. We also show that NOD and New Zealand Black mice, which have a disrupted thymic architecture and autoimmune phenotype, have lower levels of Prss16 expression compared to C57BL/6 mice. These findings support the role of Prss16 in T-cell development and susceptibility to autoimmunity in the mouse.     

Evidence against a role of gap junctions in vestibular compensation

Vestibular compensation following unilateral labyrinthectomy is associated with modifications of the membrane and firing properties of central vestibular neurons. To determine whether gap junctions could be involved in this process, immunofluorescent detection of neuronal connexin 36 and astrocytic connexin 43 was performed in the medial vestibular nucleus (MVN) of rats. In non-lesioned animals, strong staining was observed with anti-connexin 43 antibodies, while moderate staining was obtained with the anti-connexin 36 antibody. However, the expression of either type of connexin was not modified following unilateral labyrinthectomy. These morphological observations were complemented by pharmacological tests performed during extracellular recordings of MVN neurons in guinea pig brainstem slices. In non-lesioned animals, the gap junction blocker carbenoxolone reversibly decreased or suppressed the spontaneous discharge of about 60% of MVN neurons. This reduction was often associated with a long-duration disruption of the regularity of spike discharge. Both effects were mimicked by several other gap junction blockers, but not by glycyrrhizic acid, an analog of carbenoxolone that does not block gap junctions but reproduces its non-specific effects, nor by the selective inhibitor of astrocytic connexin-based networks endothelin-1. Similar effects of carbenoxolone were obtained on the spontaneous activity of ipsilesional MVN neurons recorded in brainstem slices taken from labyrinthectomized animals. Altogether, these results suggest that neuronal gap junctions are involved in shaping the spontaneous activity of MVN neurons. However, unilateral labyrinthectomy does not affect the expression of gap junctions in vestibular nuclei nor their implication in the regulation of neuronal activity.     

Oocyte maturity in relation to woman's age in in vitro fertilization cycles stimulated by single regimen

Purpose

During stimulated in vitro fertilization (IVF) cycle, up to 30% of the recovered oocytes are immature ones which have poor fertilization capacity; however, the precise influencing factors are largely unknown. Here, we analyzed the association of oocyte immaturity with woman''s age in IVF cycles stimulated by single regimen.

Materials and Methods

A total of one-hundred ninety five IVF cycles stimulated by recombinant FSH and GnRH antagonist protocol between 2003 and 2009 were analyzed retrospectively. The mean age of women was 34.2±4.0 (26-45 years). After triggering by exogenous hCG, an ultrasound-guided retrieval of oocytes was performed 35-36 hours later. All clinical data were stratified by woman''s age; group I: ≤30 (n=36), II: 31-35 (n=83), III: 36-40 (n=57), and IV: ≥41 (n=19).

Results

The total retrieved oocytes, as well as immature oocytes, were significantly lower in group IV, however, the mean % of immature oocytes was significantly higher in group IV than other age groups. Oocyte immaturity tended to decrease as increasing age in women aged 40 years or less.

Conclusion

In stimulated IVF cycle, much higher oocyte immaturity was noted in women aged 41 years or more.     

V?Gene Family Usage in Spontaneous Lymphomas of AKR Mice: Evidence for Defective Clonal Deletion

T-cell receptor (TCR) ß-chain usage and expression of the CD3, CD4, and CD8 differentiation antigens were analyzed in 14 spontaneous AKR lymphomas. Lymphoma cells massively infiltrated and/or proliferated in the organs analyzed (thymus, spleen, and mesenteric lymph nodes), giving rise to a loss of organ structure. One lymphoma occurred only in the thymus, and failed to express CD3, CD4, and CD8. All other lymphomas expressed the CD3/TCR complex. With respect to CD4 and CD8 expression, the lymphomas were either double-negative (DN), double-positive (DP), or single-positive (SP). The frequency of DP (CD4⁺8⁺) lymphomas was low compared to the frequency of DP thymocytes in a normal AKR thymus. A substantial heterogeneity was seen in the intensity of CD4 and CD8 expression among various lymphomas, which was independent of the level of CD3 expression. Considering TCR V ß gene family usage, 2 out of 14 lymphomas expressed V ß6. Normally, V ß6⁺ thymocytes are deleted from the thymocyte pool at the immature DP stage of T-cell development in AKR mice. These data support the hypothesis that the lymphocytes in the immature DP stage of T-cell development are susceptible to the induction of AKR lymphomagenesis. The presence of V ß6⁺ lymphoma cells indicates that the lymphomagenesis is accompanied by a defective clonal deletion of cells expressing a possible autoreactive TCR.     

In vitro effects of mercuric chloride (HgCl2) on human mononuclear cells

Due to the release of the toxic compounds of mercury from amalgam fillings, dental amalgam has been questioned as an adequate restoration material for tooth fillings. HgCl₂ has been found to be mitogenic for human blood lymphocytes in vitro. However, activation required much higher concentrations than are ever found in vivo. This study has been initiated to evaluate further the influence of HgCl₂ on human immunocompetent cells in vitro. It is found that HgCl₂ in a narrow concentration range has the ability to preferentially stimulate the CD4⁺ T cell subset to blast transformation and DNA synthesis. The reaction, when monitored during days 2–6, is maximal at day 6, and most blasts express the IL-2 receptor (IL-2R), indicating in vitro activation. The CD8⁺ T cell subset is not affected to the same extent. In addition, HgCl₂-induced lymphocyte reactivity is dependent on accessory cells, i.e. CD14⁺ cells.     

The Effect of Single Embryo Transfer on Perinatal Outcomes in Japan

Objective: In 2007 and 2008, the Japan Society for Reproductive Medicine and the Japan Society of Obstetrics and Gynecology issued a recommendation for single embryo transfer (SET). Thereafter, SET was implemented in 73% of in vitro fertilization (IVF) cases in Japan. The purpose of this study was to evaluate the effects of compliance with the SET recommendation on perinatal outcomes.Methods: An electronic audit of the perinatal database of the Japanese Society of Obstetrics and Gynecology was conducted from 2001 through 2010. The database comprised data of 610,726 women. Totally, 20,923 women conceived through IVF. To compare perinatal outcomes, these women were categorized into two study groups depending on whether they conceived before (2004-2005, n = 3,865) or after (2009-2010, n = 6,842) the SET recommendation statement was issued.Results: The proportion of women who conceived through IVF increased from 1.3% in 2001 to 4.8% in 2010. Compliance with the SET recommendation led to a decrease in the incidence of twin pregnancies (33.9% versus 13%, p < 0.01), incidence of preterm delivery (odds ratio [OR]: 0.54, 95% confidence interval [CI]: 0.50-0.59), low birth weight (OR: 0.42, 95% CI: 0.39-0.45), and neonatal intensive care unit admission (OR 0.70, 95% CI 0.65-0.76), but an increase in the incidence of monochorionic twins (1.6% versus 2.5%, p < 0.01).Conclusion: Compliance with the SET recommendation improved perinatal outcomes by reducing the incidence of twin pregnancies.     

Autoantibodies to double-stranded (ds)DNA immunoprecipitate 18S ribosomal RNA by virtue of their interaction with ribosomal protein S1 and suppress in vitro protein synthesis

We report that four systemic lupus erythematosus (SLE) patient sera containing anti-dsDNA antibodies, three affinity-purified anti-dsDNA IgG, and a human anti-dsDNA MoAb (33.H11) immunoprecipitate 18S ribosomal RNA from DNase-treated ³²P-labelled MOLT4 cell extract. This 18S RNA precipitation was inhibited completely by preincubating 33.H11 with calf thymus dsDNA or the recombinant human ribosomal protein S1, which was reported to cross-react with anti-dsDNA antibodies (J Immunol 1996; 156:1668–75). Whole IgG from three SLE sera with anti-dsDNA antibodies, 33.H11, and three affinity-purified anti-dsDNA IgG inhibited in vitro translation of globin mRNA (percent inhibition was 36–50%). This translation inhibition by anti-dsDNA antibodies was enhanced (67–79%) when the reticulocyte lysate was treated with DNase. Suppression of protein synthesis could be a pathogenic mechanism of anti-dsDNA antibodies, since it has also been shown that anti-dsDNA penetrates living cells (J Immunol 1995; 154:4857–64) in culture.     

In vitro evidence of possible influence of scutellarein towards bile acids' metabolism

Background

The glucuronidation process has been regarded as the key elimination process for toxic bile acids. UDP-glucuronosyltransferase (UGT) 1A3 is one important metabolizing enzyme involved in this process.

Objective

To evaluate the inhibition of UGT1A3 by scutellarein which is an important herbal ingredient using in vitro method, trying to indicate the possibility of toxicity due to the accumulation of toxic bile acids.

Methods

Due to the difficulty to gain the standards of biles acids'' glucuronides, the recombinant UGT1A3-catalyzed 4-methylumbelliferone (4-MU) glucuronidation reaction was employed to profile the activity of UGT1A3.

Results

The results showed that scutellarein inhibited UGT1A3 in a concentration-dependent behaviour. Competitive inhibition was demonstrated using both Dixon plot and Lineweaver-Burk plot, and the inhibition kinetic parameter (K_i) was calculated to be 5.8uM.

Conclusion

All these data reminded the necessary monitoring of the levels of bile acids in plasma when utilizing scutellarein and the herbs containing this compound.     

In Vitro Evaluation of Antiplasmodial Activity of Extracts of Acanthospermum Hispidum DC (Asteraceae) and Ficus Thonningii Blume (Moraceae), Two Plants Used in Traditional Medicine in the Republic of Congo

The aim of this study was to evaluate extracts from two medicinal plants, Acanthospermum hispidum and Ficus thonningii, used in traditional medicine in Congo Brazzaville, for in vitro antiplasmodial activities against two laboratory strains of Plasmodium falciparum: the chloroquine sensitive 3D7 and the chloroquine resistant Dd2. ELISA HRP2 assay was used to evaluate the in vitro inhibitory activity of the extracts alone or in combination with chloroquine. Cytotoxicity was assessed on human HeLa cell line and reflected by the selectivity index. Methanolic extract of Acanthospermum hispidum exhibited a strong and a moderate inhibitory activity on the growth of Dd2 and 3D7 at 2.8 µg/ml and 9.2 µg/ml concentrations respectively with a selectivity index >10. The combination of the most active extract (methanolic extract of Acanthospermum hispidum) with chloroquine showed a synergistic interaction on both strains. The good selectivity index of Acanthospermum hispidum on HeLa cells reflects the safety of this plant. Extracts from Ficus thonningii did not show any promising antiplasmodial activity on both 3D7 and Dd2. Except the methanolic extract which exhibited a slight antiplasmodial activity with inhibitory concentration and selectivity index corresponding to 9.61 µg/ml and 11.16 respectively. Methanolic extract of Acanthospermum hispidum exhibited moderate to high inhibitory activity on 3D7 and Dd2 laboratory strains and a synergistic antimalarial effect when combined with chloroquine. Ficus thonningii seems to have no antimalarial activity. Phytochemical analysis, in vivo investigations using animal models and later clinical trials in collaboration with traditional practitioners are necessary to clarify the potential antimalarial activity of both plants.     

Qualitative Phytochemical Screening and In Vitro Antimicrobial Effects of Methanol Stem Bark Extract of Ficus Thonningii (Moraceae)

The methanolic stem bark extract of Ficus thonningii (Moraceae) was subjected to preliminary phytochemical screening and in vitro antimicrobial tests. The phytochemical tests was carried out using standard methods of analysis and these investigations revealed the presence of alkaloids, anthraquinones, carbohydrates, flavonoids, saponins and tannins. The antimicrobial activity of the plant extract was assayed using the agar plate disc diffusion and nutrient broth dilution techniques. Test micro organisms were: Escherichia coli, Klebsiella spp, Pseudomonas aeruginosa, Salmonella typhi (Gram-negative), Staphylococcus aureus and Streptococcus spp. (Gram-positive). The extracts inhibited the growth of all the test organisms at different concentrations especially against Pseudomonas aeruginosa and Streptococcus spp. which had mean inhibition zone of 33.33±7.33 mm and 32.33±2.51 mm respectively. The results showed the MIC of 10 mg ml⁻¹ against pseudomonas and 1.25 against remaining organisms tested. The MBC against Staphylococcus aureus was 2.5 mg ml⁻¹ and that of Streptococcus spp. was found to be 0.625mg ml⁻¹. The extracts showed varied inhibitory activity against the organisms studied.     

Advances in 3D cell culture technologies enabling tissue‐like structures to be created in vitro

Research in mammalian cell biology often relies on developing in vitro models to enable the growth of cells in the laboratory to investigate a specific biological mechanism or process under different test conditions. The quality of such models and how they represent the behavior of cells in real tissues plays a critical role in the value of the data produced and how it is used. It is particularly important to recognize how the structure of a cell influences its function and how co‐culture models can be used to more closely represent the structure of real tissue. In recent years, technologies have been developed to enhance the way in which researchers can grow cells and more readily create tissue‐like structures. Here we identify the limitations of culturing mammalian cells by conventional methods on two‐dimensional (2D) substrates and review the popular approaches currently available that enable the development of three‐dimensional (3D) tissue models in vitro. There are now many ways in which the growth environment for cultured cells can be altered to encourage 3D cell growth. Approaches to 3D culture can be broadly categorized into scaffold‐free or scaffold‐based culture systems, with scaffolds made from either natural or synthetic materials. There is no one particular solution that currently satisfies all requirements and researchers must select the appropriate method in line with their needs. Using such technology in conjunction with other modern resources in cell biology (e.g. human stem cells) will provide new opportunities to create robust human tissue mimetics for use in basic research and drug discovery. Application of such models will contribute to advancing basic research, increasing the predictive accuracy of compounds, and reducing animal usage in biomedical science.     

Antimicrobial Activity of Extracts from In Vivo and In Vitro Propagated Lamium Album L. Plants

The antimicrobial activity of 18 different extracts from in vivo and in vitro grown L. album L. plants was evaluated against clinical bacteria and yeasts using the well diffusion method. All the used extracts demonstrated antibacterial activity, whereas only the water extracts from leaves (in vivo) possessed antifungal activity against Candida albicans NBIMCC 72 and Candida glabrata NBIMCC 8673 (14 and 20 mm diameter of inhibition zones and MIC 10 mg/ml, respectively). The methanol and ethanol extracts obtained from the in vitro propagated plants had a broader spectrum of antibacterial activity than those from in vivo plants, while the opposite tendency was observed for the chloroform extracts. All tested flower extracts possessed antimicrobial activity. The chloroform extract from in vivo flowers demonstrated the highest activity against E. faecalis NBIMCC 3915, S. aureus NBIMCC 3703, P. hauseri NBIMCC 1339 and P. aeruginosa NBIMCC 3700 (22 mm, 13 mm, 11 mm, 23 mm zone diameter of inhibition and MIC 0.313 mg/ml, respectively). The water extracts from leaves (both in vivo and in vitro) possessed higher antibacterial activity than extract from flowers. The obtained results showed that both in vivo and in vitro propagated L. album L. could be used as a source of antibacterial substances.     

Multiple meiotic errors caused by predivision of chromatids in women of advanced maternal age undergoing in vitro fertilisation

Chromosome aneuploidy is a major cause of pregnancy loss, abnormal pregnancy and live births following both natural conception and in vitro fertilisation (IVF) and increases exponentially with maternal age in the decade preceding the menopause. Molecular genetic analysis following natural conception and spontaneous miscarriage demonstrates that trisomies arise mainly in female meiosis and particularly in the first meiotic division. Here, we studied copy number gains and losses for all chromosomes in the two by-products of female meiosis, the first and second polar bodies, and the corresponding zygotes in women of advanced maternal age undergoing IVF, using microarray comparative genomic hybridisation (array CGH). Analysis of the segregation patterns underlying the copy number changes reveals that premature predivision of chromatids rather than non-disjunction of whole chromosomes causes almost all errors in the first meiotic division and unlike natural conception, over half of aneuploidies result from errors in the second meiotic division. Furthermore, most abnormal zygotes had multiple aneuploidies. These differences in the aetiology of aneuploidy in IVF compared with natural conception may indicate a role for ovarian stimulation in perturbing meiosis in ageing oocytes.     

Models of Respiratory Infections: Virus-Induced Asthma Exacerbations and Beyond

Respiratory infections are one of the main health problems worldwide. They are a challenging field of study due to an intricate relationship between the pathogenicity of microbes and the host''s defenses. To better understand mechanisms of respiratory infections, different models have been developed. A model is the reproduction of a disease in a system that mimics human pathophysiology. For this reason, the best models should closely resemble real-life conditions. Thus, the human model is the best. However, human models of respiratory infections have some disadvantages that limit their role. Therefore, other models, including animal, in vitro, and mathematical ones, have been developed. We will discuss advantages and limitations of available models and focus on models of viral infections as triggers of asthma exacerbations, viral infections being one of the most frequent causes of exacerbating disease. Future studies should focus on the interrelation of various models.