Genetics and imaging to assess oocyte and preimplantation embryo health.

Two major criteria are currently used in human assisted reproductive technologies (ART) to evaluate oocyte and preimplantation embryo health: (1) rate of preimplantation embryonic development; and (2) overall morphology. A major gene that regulates the rate of preimplantation development is the preimplantation embryo development (Ped) gene, discovered in our laboratory. In mice, presence of the Ped gene product, Qa-2 protein, results in a fast rate of preimplantation embryonic development, compared with a slow rate of preimplantation embryonic development for embryos that are lacking Qa-2 protein. Moreover, mice that express Qa-2 protein have an overall reproductive advantage that extends beyond the preimplantation period, including higher survival to birth, higher birthweight, and higher survival to weaning. Data are presented that suggest that Qa-2 increases the rate of development of early embryos by acting as a cell-signalling molecule and that phosphatidylinositol-32 kinase is involved in the cell-signalling pathway. The most likely human homologue of Qa-2 has recently been identified as human leukocyte antigen (HLA)-G. Data are presented which show that HLA-G, like Qa-2, is located in lipid rafts, implying that HLA-G also acts as a signalling molecule. In order to better evaluate the second criterion used in ART (i.e. overall morphology), a unique and innovative imaging microscope has been constructed, the Keck 3-D fusion microscope (Keck 3DFM). The Keck 3DFM combines five different microscopic modes into a single platform, allowing multi-modal imaging of the specimen. One of the modes, the quadrature tomographic microscope (QTM), creates digital images of non-stained transparent cells by measuring changes in the index of refraction. Quadrature tomographic microscope images of oocytes and preimplantation mouse embryos are presented for the first time. The digital information from the QTM images should allow the number of cells in a preimplantation embryo to be counted non-invasively. The Keck 3DFM is also being used to assess mitochondrial distribution in mouse oocytes and embryos by using the k-means clustering algorithm. Both the number of cells in preimplantation embryos and mitochondrial distribution are related to oocyte and embryo health. New imaging data obtained from the Keck 3DFM, combined with genetic and biochemical approaches, have the promise of being able to distinguish healthy from unhealthy oocytes and embryos in a non-invasive manner. The goal is to apply the information from our mouse model system to the clinic in order to identify one and only one healthy embryo for transfer back to the mother undergoing an ART procedure. This approach has the potential to increase the success rate of ART and to decrease the high, and undesirable, multiple birth rate presently associated with ART.

Molecular genetics of ulcerative colitis-associated colon cancer in the interleukin 2- and beta(2)-microglobulin-deficient mouse.

Mice deficient in beta(2)-microglobulin and interleukin 2 (beta(2)m(null) x IL-2(null)) spontaneously develop colon cancer in the setting of chronic ulcerative colitis (UC). We investigated mutations of the Apc and p53 genes and microsatellite instability in colonic adenocarcinomas arising in this model. Mutations of the Apc and p53 genes in the regions corresponding to mutation hot spots in human colorectal cancer were determined by sequencing in 11 colonic adenocarcinomas. Microsatellite instability was determined in matched normal and neoplastic DNA at five loci. All 11 adenocarcinomas harbored Apc mutations. Of these 11 tumors, 5 harbored truncating mutations. A total of 67 Apc mutations were found in these 11 tumors; 59 were missense mutations, whereas 8 were frameshift or nonsense mutations. Six of the 11 adenocarcinomas harbored p53 mutations. A total of seven p53 mutations were found in these 11 tumors; all mutations were transitions, 4 of which were C:G-->T:A transitions occurring in codon 229 at cytosine-guanine dinucleotides. Nine adenocarcinomas exhibited microsatellite instability in at least one of the five loci examined; 1 tumor had microsatellite instability in two loci. Molecular genetics, as well as clinical features, of colon cancer in the beta(2)m(null) x IL-2(null) mice are similar to those of human UC-associated colorectal cancer. As such, this model appears to be an excellent animal model to study UC-associated colorectal carcinogenesis.

Chronic murine colitis is dependent on the CD154/CD40 pathway and can be attenuated by anti-CD154 administration.

BACKGROUND & AIMS: Experimental colitis in most animal models is caused by dysregulation of T lymphocytes that display a T helper 1 (Th1) phenotype. CD154 (CD40L/gp39), a member of the tumor necrosis factor (TNF) family, is up-regulated on T cells on activation and has been shown to play a key role in the induction of a Th1 response. We investigated whether chronic experimental colitis is dependent on the CD154/CD40 pathway and whether disease can be prevented by anti-CD154 antibody treatment. METHODS: Two models of chronic colitis were used: CD45Rb(hi) cell transfer into recombination activation gene-deficient (Rag(-/-)) mice and bone marrow transplant of tgepsilon26 animals. In both models, mice were reconstituted with cells from CD154-deficient animals. In another series of experiments, wild-type CD45Rb(hi) T cell-reconstituted recipients were treated with anti-CD154, either from the start of the experiment or after onset of disease. RESULTS: T cells deficient in CD154 induced a milder clinical disease, less weight loss, and fewer histologic signs of colitis than wild-type cells. The level of interleukin 12 in the serum of CD154-deficient T-cell recipients was 5-fold less than that of wild-type cell recipients. Nevertheless, no signs of deviation from a Th1 phenotype were observed. Treatment with anti-CD154 antibodies substantially impaired disease development, even when started after the onset of colitis. CONCLUSIONS: The CD154/CD40 pathway plays a critical role in Th1-induced chronic experimental colitis. Blocking CD154, even after the onset of disease, ameliorates colitis but does not induce a T helper 2 (Th2) phenotype.

Both the lymphotoxin and tumor necrosis factor pathways are involved in experimental murine models of colitis.

BACKGROUND & AIMS: Membrane lymphotoxin (LT) alpha/beta, a member of the tumor necrosis factor (TNF) family of immune regulatory molecules, is involved both in the development of secondary lymphoid tissues and the maintenance of organized lymphoid tissues in the adult. Defects observed in the mucosal immune system in animals with a genetically disrupted LTalpha/beta pathway coupled with the expression of LTalpha/beta in activated T cells motivated an examination of the importance of this pathway in experimental colitis. METHODS: Soluble LTbeta receptor (LTbetaR) immunoglobulin fusion protein was used to inhibit the LTalpha/beta/light axis in two independent rodent models of colitis: CD45RBhi CD4(+)-reconstituted SCID mice and bone marrow-transplanted tg26 mice (BM --> tg26). RESULTS: Treatment with LTbetaR immunoglobulin attenuated the development of both the clinical and histological manifestations of the disease in these two murine models of colitis. Given the success of TNF inhibitors in the treatment of human Crohn's disease, the effects of LTbetaR immunoglobulin have been compared with antibody to TNF in the BM --> tg26 model, and both treatments were equally efficacious. CONCLUSIONS: The LT pathway plays a role in the development of colitis as important as that of the TNF system and, therefore, represents a potential novel intervention point for the treatment of inflammatory bowel disease.

Development of colonic adenocarcinomas in a mouse model of ulcerative colitis.

Mice deficient in both interleukin-2 and beta 2-microglobulin expression (Beta 2mullnull x IL-2null mice) develop an inflammatory disease of the colon resembling ulcerative colitis. To examine long-term complications of disease in these mice, a group of 34 Beta 2mnull x IL-2null mice was monitored for 6-12 months. Development of clinical disease was assessed by wasting, general appearance, and diarrhea. Further analysis included histologic examination of the distal colon for colitis, staining of CD4+ T cells for surface activation markers, and cytoplasmic staining of CD4+ T cells for IFN-gamma and TNF-alpha. These older Beta 2mnull x IL-2null mice had activated CD4+ T cells as assessed by surface markers on flow cytometry. Cytoplasmic staining revealed IFN-gamma production, but not TNF-alpha production by CD4+ T cells. The majority of these older Beta 2mnull x IL-2null mice continued to have colitis on histology. However, they lived much longer and had less wasting in comparison to IL-2null mice. At necropsy, 11 (32%) of 34 of the Beta 2mnull x IL-2null mice had tumors in the proximal half of the colon. Histologic examination confirmed these tumors to be adenocarcinomas. These mice may be useful as a model for studying carcinogenesis in chronic colitis.

Consequences of Fas-ligand and perforin expression by colon T cells in a mouse model of inflammatory bowel disease.

BACKGROUND & AIMS: We describe a type of colitis that develops after transplantation of nonallogeneic wt bone marrow cells into T cell- and natural killer cell-deficient Tg26 mice (BM-->Tg26). In these animals, severe wasting and inflammation of the colon correlates with the expansion of mucosal T lymphocytes that displays cytotoxic activity. The aims of this study were to determine the relative contribution of perforin and Fas ligand (Fas-L) expression to the cytotoxic action of these T cells and to examine the influence of each pathway in this model of colitis. METHODS: Colonic T cells were tested for their ability to mediate Fas- and perforin-dependent killing in redirected cytotoxicity assays. Bone marrow cells from donor mice lacking either Fas-L (gld mice) or perforin (PFPnull mice) or both molecules were used to reconstitute Tg26 mice. RESULTS: Colon cytotoxic T lymphocyte displayed both Fas- and perforin-dependent killing. Deficiency in perforin, but not Fas-L, resulted in reduced incidence of wasting and, to a lesser extent, severe colitis in BM-->Tg26 animals. CONCLUSIONS: Colon T cells from BM-->Tg26 mice express both perforin and Fas-L. Although neither pathway is critical in the development of colitis, perforin does have a measurable influence on disease in the BM-->Tg26 colitis model.

T cell-mediated pathology in two models of experimental colitis depends predominantly on the interleukin 12/Signal transducer and activator of transcription (Stat)-4 pathway, but is not conditional on interferon gamma expression by T cells.

The requirements for interleukin (IL)-12/signal transducer and activator of transcription (Stat)-4 signaling and induction of T cell-specific interferon (IFN)-gamma expression in the development of T helper cell (Th)1-type pathology were examined in two different models of experimental colitis. In each model, abnormal reconstitution of the T cell compartment in immunodeficient mice by adoptive cell transfer leads to a wasting syndrome and inflammation of the colon, induced by IFN-gamma and tumor necrosis factor (TNF)-alpha-producing T cells. We show here that treatment with anti-IL-12 antibodies in one of the models, or reconstitution with T cells from Stat-4-deficient (Stat-4(null)) mice in both models resulted in a milder disease in the majority of recipient animals, compared with those that were left untreated or that had been reconstituted with wt cells. Protected mice in each group also harbored lower frequencies of IFN-gamma-producing T cells than did diseased mice, suggesting that effects on wasting and colitis resulted from the attenuation of IFN-gamma expression by T cells. To test whether the development of pathogenic T cells in the two colitis models was directly dependent on T cell-specific IFN-gamma expression, IFN-gammanull donors were used for T cell reconstitution in each system. Surprisingly, large numbers of IFN-gammanull-reconstituted mice developed wasting and colitis, which in many cases was of comparable severity to that seen in animals reconstituted with wt cells. Furthermore, T cells from these animals expressed TNF-alpha, demonstrating that they had retained the ability to produce another proinflammatory cytokine. Taken together, these results demonstrate that in some forms of chronic experimental colitis the development of pathogenic T cells is influenced predominantly, though not exclusively, by IL-12 via the actions of Stat-4 proteins. Furthermore, our data suggest that in the models of colitis studied here the effects of IL-12/Stat-4 or other Th1 promoting pathways are not limited to the induction of IFN-gamma gene expression in T lymphocytes.