Search

Neoadjuvant intratumoral (IT) therapy could amplify the weak responses to checkpoint blockade therapy observed in breast cancer (BC). In this study, we administered neoadjuvant IT anti-canine PD-1 therapy (IT acPD-1) alone or combined with IT cowpea mosaic virus therapy (IT CPMV/acPD-1) to companion dogs diagnosed with canine mammary cancer (CMC), a spontaneous tumor resembling human BC. CMC patients treated weekly with acPD-1 (n = 3) or CPMV/acPD-1 (n = 3) for four weeks or with CPMV/acPD-1 (n = 3 patients not candidates for surgery) for up to 11 weeks did not experience immune-related adverse events. We found that acPD-1 and CPMV/acPD-1 injections resulted in tumor control and a reduction in injected tumors in all patients and in noninjected tumors located in the ipsilateral and contralateral mammary chains of treated dogs. In two metastatic CMC patients, CPMV/acPD-1 treatments resulted in the control and reduction of established lung metastases. CPMV/acPD-1 treatments were associated with altered gene expression related to TLR1-4 signaling and complement pathways. These novel therapies could be effective for CMC patients. Owing to the extensive similarities between CMC and human BC, IT CPMV combined with approved anti-PD-1 therapies could be a novel and effective immunotherapy to treat local BC and suppress metastatic BC.

Lactate is a key metabolite for the normal occurrence of spermatogenesis. In the testis, lactate is produced by the Sertoli cells and transported to germline cells. Monocarboxylate transporters (MCTs) are key players in that process. Among the family of MCTs, MCT1 is at least partly responsible for lactate uptake by the germ cells. We aimed to perform a first assessment of the role of MCT1 in male reproductive potential. Mct1 conditional knockout (cKO) mice were used for morphometric evaluation, testicular morphology, and sperm parameter assessment. Serum steroid hormones levels were also measured. cKO animals showed a decrease in gonadosomatic index, testis weight, and seminiferous tubular diameters. Deletion of MCT1 also causes morphological changes in the organization of the seminiferous tubules and on Sertoli cell morphology. These changes resulted in failure of spermatogenesis with depletion of germ cells and total absence of spermatozoa. MCT1 cKO animals presented also hormonal dysregulation, with a decrease in serum 17β-estradiol levels. In conclusion, MCT1 is pivotal for male reproductive potential. Absence of MCT1 results in maintenance of undifferentiated spermatogonia pool and compromised sperm production.

Thyroid carcinomas that harbor RET/PTC oncogenes are well differentiated, relatively benign neoplasms compared with those expressing oncogenic RAS or BRAF mutations despite signaling through shared transforming pathways. A distinction, however, is that RET/PTCs induce immunostimulatory programs, suggesting that, in the case of this tumor type, the additional pro-inflammatory pathway reduces aggressiveness. Here, we demonstrate that pro-inflammatory programs are selectively activated by TRAF2 and TRAF6 association with RET/PTC oncoproteins. Eliminating this mechanism reduces pro-inflammatory cytokine production without decreasing transformation efficiency. Conversely, ablating MEK/ERK or PI3K/AKT signaling eliminates transformation but not pro-inflammatory cytokine secretion. Functional uncoupling of the two pathways demonstrates that intrinsic pro-inflammatory pathways are not required for cellular transformation and suggests a need for further investigation into the role inflammation plays in thyroid tumor progression.

The oncogenic fusion protein RET/PTC3 (RP3) that is expressed in papillary thyroid carcinoma (PTC) and thyroid epithelia in Hashimoto's thyroiditis activates nuclear factor-kappa B (NF-κB) and induces pro-inflammatory gene expression; however, the mechanism of this activation is unknown. To address this, we expressed RP3 in murine embryonic fibroblasts (MEFs) lacking key classical and noncanonical NF-κB signaling components. In wild-type MEFs, RP3 upregulated CCL2, CXCL1, granulocyte-macrophage colony-stimulating factor and tumor necrosis factor expression and activated classical but not noncanonical NF-κB. RP3-activated NF-κB in IκB kinase (IKK)β(-/-) MEFs but not IKKα- or NF-κB essential modulator (NEMO)-deficient cells and activation was inhibited by a peptide that blocks NEMO binding to the IKKs. RP3 increased the levels of NF-κB-inducing kinase (NIK) and did not activate NF-κB in NIK-deficient MEFs. Notably, NIK stabilization was not accompanied by TRAF3 degradation demonstrating that RP3 disrupts normal basal NIK regulation. Dominant-negative NIK blocked RP3-induced NF-κB activation and an RP3 signaling mutant (RP3(Y588F)) did not stabilize NIK. Finally, examination of PTC specimens revealed strong positive staining for NIK. We therefore conclude that RP3 activates classical NF-κB via NIK, NEMO and IKKα. Importantly, our findings reveal a novel mechanism for oncogene-induced NF-κB activation via stabilization of NIK.

Impairment of the p27(kip1) function, caused by a drastic reduction of its expression or cytoplasmic mislocalization, has been frequently observed in thyroid carcinomas. To understand the role of p27(kip1) impairment in thyroid carcinogenesis, we investigated the consequences of the loss of p27(kip1) expression in the context of a mouse modeling of papillary thyroid cancer, expressing the TRK-T1 oncogene under the transcriptional control of thyroglobulin promoter. We found that double mutant mice homozygous for a p27(kip1) null allele (TRK-T1/p27(-/-)) display a higher incidence of papillary thyroid carcinomas, with a shorter latency period and increased proliferation index, compared with p27(kip1) wild-type compounds (TRK-T1/p27(+/+)). Consistently, double mutant mice heterozygous for a p27(kip1) null allele (TRK-T1/p27(+/-)) show an incidence of thyroid carcinomas that is intermediate between TRK-T1/p27(-/-) and TRK-T1/p27(+/+) mice. Therefore, our findings suggest a dose-dependent role of p27(kip1) function in papillary thyroid cancer development.

The RET/PTC3 (RP3) fusion protein is an oncogene expressed during the development of thyroid cancer and in thyroid epithelial cells of patients with Hashimoto's thyroiditis. RP3 has two immunological properties: 1) it encodes a chimeric protein including peptides that may be targets of antitumor immune responses and 2) it is a tyrosine kinase that can activate NF-kappaB transcriptional programs, induce secretion of proinflammatory mediators, and stimulate innate immunity. To distinguish the antigenic properties of the RP3 oncoprotein from its signaling function, a transplantable tumor system was developed. Tumors expressing the functional, but not mutant, form of RP3 show enhanced infiltration of CD8(+) lymphocytes, myeloid-derived CD11b(+)Gr1(+) cells, and enhanced growth in immunocompetent mice. In contrast, RP3 signaling mutant-expressing tumors maintained enhanced infiltration of CD8(+) lymphocytes did not enhance recruitment of CD11b(+)Gr1(+) cells and showed a decreased tumor incidence. These results implicate a role for RP3 function in enhancing a tumor-suppressive innate inflammatory response. These experiments support a mechanism whereby oncogenes can directly recruit and activate innate and adaptive immune cells, resulting in enhanced tumor progression.

Background: One of the greatest impediments to cancer immunotherapy is the paucity of antigens that are tumor specific, sufficiently immunogenic, and shared among patients. Mucosa-restricted antigens that are expressed by tumor cells represent a novel class of vaccine targets that are characterized by immunologic privilege, which limits systemic tolerance to those antigens, and immunologic partitioning, which shields mucosae from systemic autoimmune responses. Here we defined the immunogenicity and antitumor efficacy of guanylyl cyclase C (GCC), a protein that is normally restricted to intestinal mucosa and universally expressed by metastatic colorectal cancer., Methods: BALB/c mice (n = 197) were immunized with recombinant GCC-expressing viral vectors before (prophylactic) or after (therapeutic) a lethal challenge of GCC-expressing mouse colon cancer cells, and antitumor efficacy was monitored by quantifying metastasis and survival. Induction of autoimmunity was monitored by histopathology. Induction of GCC-specific B-cell and CD4(+) and CD8(+) T-cell responses were determined by enzyme-linked immunosorbent assay and ELISpot, respectively. Tolerance to GCC was quantified by comparing responses in GCC-deficient (n = 45) and wild-type (n = 69) C57BL/6 mice. Statistical tests were two-sided., Results: Immunization with GCC-expressing viral vectors reduced the formation of metastases to liver (control vs GCC: mean = 30.4 vs 3.55 nodules, difference = 26.9 nodules, 95% confidence interval [CI] = 8.47 to 45.3 nodules; P = .008) and lung (control vs GCC: mean = 263 vs 55.7 nodules, difference = 207, 95% CI = 163 to 251; P < .001) and extended the median survival of mice with established lung metastases following therapeutic immunization (control vs GCC: 29 vs 38 days, P = .024), without autoimmunity. Antitumor efficacy reflected asymmetrical tolerance that was characterized by CD8(+) T-cell, but not CD4(+) T-cell or antibody, responses., Conclusions: Immunologic partitioning together with immunologic privilege highlight the potential of mucosa-restricted antigens, particularly GCC, as therapeutic targets for metastatic cancer.

Background: Farnesyltransferase inhibitors (FTI) are small molecule agents originally formulated to inhibit the oncogenic functions of Ras. Although subsequent analysis of FTI activity revealed wider effects on other pathways, the drug has been demonstrated to reduce Ras signaling by direct measurements. The purpose of the current study was to determine if FTI could be used to inhibit the inflammatory activities of a known Ras-activating human oncoprotein, RET/PTC3. RET/PTC3 is a fusion oncoprotein expressed in the thyroid epithelium of patients afflicted with thyroid autoimmune disease and/or differentiated thyroid carcinoma. Previous studies have demonstrated that RET/PTC3 signals through Ras and can provoke nuclear translocation of NFkappaB and the downstream release of pro-inflammatory mediators from thyroid follicular cells in vitro and in vivo, making it an ideal target for studies using FTI., Methods: For the studies described here, an in vitro assay was developed to measure FTI inhibition of RET/PTC3 pro-inflammatory effects. Rat thyrocytes transfected with RET/PTC3 or vector control cDNA were co-cultured with FTI and examined for inhibition of chemokine expression and secretion measured by RT-PCR and ELISA. Immunoblot analysis was used to confirm the level at which FTI acts on RET/PTC3-expressing cells, and Annexin V/PI staining of cells was used to assess cell death in RET/PTC3-expressing cells co-cultured with FTI., Results: These analyses revealed significant mRNA and protein inhibition of chemokines Ccl2 and Cxcl1 with nanomolar doses of FTI. Neither RET/PTC3 protein expression nor apoptosis were affected at any dose of FTI investigated., Conclusion: These data suggest that FTI may be applied as an effective inhibitor for RET/PTC3-oncogene induced pro-inflammatory mediators.

Colorectal cancer is a leading cause of cancer-related mortality worldwide. Surgery and chemoradiation exhibit incomplete efficacy and, ultimately, 50% of patients die of metastatic disease. In the context of that unmet clinical need, immunotherapeutic approaches have enjoyed limited success, partly because of a paucity of suitable antigen targets. However, exploitation of immune compartmentalization, employing antigens with expression restricted to normal intestinal mucosa and derivative colorectal tumors--cancer mucosa antigens (CMAs)--may represent a previously unrecognized class of immune targets supporting efficacious antitumor immunotherapy. Guanylyl cyclase C (GCC) is an intestine/colorectal cancer-restricted protein ideally suited as the first CMA for clinical evaluation.

Colorectal cancer is the third most common malignancy and the second most common cause of cancer-related mortality worldwide. While surgery remains the mainstay of therapy, approximately 50% of persons who undergo resection develop parenchymal metastatic disease. Unfortunately, current therapeutic regimens offer little improvement in survival. Using immunotherapy to fill this therapeutic gap has enjoyed limited success, reflecting a paucity of tumor-associated antigens. In that context, there is a significant unrealized opportunity to exploit structural and functional immune system compartmentalization to generate a therapeutic immune response against metastatic colorectal tumors employing biomarkers whose expression is normally confined to intestinal epithelial cells and their derivative malignancies. This novel class of biomarkers, here termed cancer mucosa antigens, may fill the unmet therapeutic need for colorectal cancer-associated immune targets. As a concrete example, guanylyl cyclase C is an intestinal mucosa-specific biomarker ideally suited to test this hypothesis and serve as the first cancer mucosa antigen for colorectal cancer immunotherapy. Here, we discuss colorectal cancer immunity, immune compartmentalization and preliminary results targeting guanylyl cyclase C in mouse models of colorectal cancer, as well as the potential paradigm shift to employing cancer mucosa antigens in immunotherapy of colorectal cancer.

Many models exist to explain the induction and perpetuation of autoimmune diseases. Despite their validation in a variety of animal models, the basis for autoimmune disease in humans remains unknown. Here, we propose that an important aspect of autoimmune disease is the active participation of the target organ due to endogenously produced co-stimulatory factors that cause prolonged antigen presentation and lymphocyte activation. Evidence suggests that a major source of such endogenous signaling comes from newly transformed cells within the target organ that produce pro-inflammatory factors.

Ras oncogenes are frequently mutated in thyroid carcinomas. To verify the role played by N-ras in thyroid carcinogenesis, we generated transgenic mice in which a human N-ras(Gln61Lys) oncogene (Tg-N-ras) was expressed in the thyroid follicular cells. Tg-N-ras mice developed thyroid follicular neoplasms; 11% developed follicular adenomas and approximately 40% developed invasive follicular carcinomas, in some cases with a mixed papillary/follicular morphology. About 25% of the Tg-N-ras carcinomas displayed large, poorly differentiated areas, featuring vascular invasion and forming lung, bone or liver distant metastases. N-ras(Gln61Lys) expression in cultured PC Cl 3 thyrocytes induced thyroid-stimulating hormone-independent proliferation and genomic instability with micronuclei formation and centrosome amplification. These findings support the notion that mutated ras oncogenes could be able to drive the formation of thyroid tumors that can progress to poorly differentiated, metastatic carcinomas.

The myeloid progenitor cell compartment (MPC) exhibits pronounced expansion in human myeloid leukemias. It is becoming more apparent that progression of myelodysplastic syndromes and myeloproliferative diseases to acute myelogenous leukemia is the result of defects in progenitor cell maturation. The MPC of bone marrow was analyzed in mice using a cell culture assay for measuring the relative frequency of proliferative myeloid progenitors. Response to the cytokines SCF, IL-3, and GM-CSF was determined by this assay for the leukemic mouse strain BXH-2 and ten other inbred mouse strains. Significant differences were found to exist among ten inbred mouse strains in the nature of their MPC in bone marrow, indicating the presence of genetic polymorphisms responsible for the divergence. The SWR/J and FVB/J strains show consistently low frequencies of myeloid progenitors, while the DBA/2J and SJL/J inbred strains show consistently high frequencies of myeloid progenitors within the bone marrow compartment. In addition, in silico linkage disequilibrium analysis was conducted to identify possible chromosomal regions responsible for the phenotypic variation. Given the importance of this cell compartment in leukemia progression and the soon to be released genomic sequence of 15 mouse strains, these differences may provide a valuable tool for research into leukemia.

Context: RET/papillary thyroid cancer (PTC) is a marker for papillary thyroid carcinoma, but its specificity has been questioned because of the disputed identification of RET/PTC in Hashimoto's thyroiditis (HT), oncocytic tumors, and other thyroid lesions., Objective: The objective of this study was to determine 1) whether RET/PTC occurs in nonneoplastic follicular cells of HT, and 2) its recombination rate in thyroid tumors., Design/patients: Forty-three samples from 31 cases of HT were examined using interphase fluorescence in situ hybridization (FISH) with RET probes spanning the breakpoint region; real-time RT-PCR to quantify RET/PTC1, RET/PTC3, and c-RET transcripts; and RT-PCR after laser capture microdissection to enrich samples for follicular cells. The results were compared with those similarly obtained in 34 papillary carcinomas, eight thyroid oncocytic tumors, and 21 normal thyroids., Results: Normal samples showed no RET rearrangement. Sixty-eight percent (15 of 22) of HT were positive by FISH; in all thyroiditis, signals were localized to rare nonneoplastic follicular cells; low-level RET/PTC was identified in 17% (five of 29) of thyroiditis cases by real-time RT-PCR and in an additional six of 11 real-time negative cases after increasing sensitivity with laser capture microdissection. Low RET/PTC1 levels were detected in 26% (nine of 34) of papillary carcinomas with an expression pattern and proportion of FISH-positive cells similar to those of the thyroiditis. Forty-seven percent (16 of 34) of papillary carcinomas and one oncocytic carcinoma expressed high RET/PTC1 mRNA levels., Conclusions: Low-level RET/PTC recombination occurs in nonneoplastic follicular cells in HT and in a subset of papillary thyroid carcinomas. RET/PTC expression variability should be taken into account for the molecular diagnosis of thyroid lesions. Overlapping molecular mechanisms may govern early stages of tumor development and inflammation in the thyroid.

The activation of RET proto-oncogene through chromosomal translocation is reported as being unique to papillary thyroid carcinomas. However, the reported prevalence of RET/PTC activation in papillary carcinoma was variable, and the clinical relevance of RET/PTC rearrangements in papillary carcinomas is still controversial. To investigate the roles of RET rearrangement in the carcinogenesis of papillary thyroid carcinoma, we have studied RET activation and p53 overexpression in various thyroid lesions of the Japanese population by immunohistochemical technique. RET activation and p53 overexpression were studied in 40 papillary carcinomas, 6 poorly differentiated carcinomas, 4 undifferentiated carcinomas, 2 medullary carcinomas, 2 follicular carcinomas and 19 follicular adenomas. RET activation was observed in 12 out of 40 papillary carcinomas, while no immunoreactivity of RET was detected in other lesions. P53 overexpression was observed in only 1 of 40 papillary carcinomas, but in 2 poorly differentiated carcinomas and 4 undifferentiated carcinomas. The prevalence of RET/PTC activation in papillary carcinoma among the Japanese population was higher than in previous reports. Immunohistochemical technique is proved to be a useful tool to detect RFT/PTC activation in thyroid tumors. RET rearrangements are restricted to a well-differentiated papillary carcinoma, suggesting that RET/PTC positive papillary carcinomas do not progress to undifferentiated carcinoma.

Thyroid cancers, like hematological malignancies, are commonly associated with chromosomal translocations leading to the formation of fusion proteins. Through altered signaling by fusion proteins, cell death and survival pathways are disrupted and the physiological balance of cell-cell communication may be lost. A consequence of this disruption is the release of factors by stressed cells that alert the host. One type of host response is leukocytic infiltration that may develop into chronic inflammation or autoimmune disease. Although inflammation can be associated with neoplastic tissue, the mechanism driving this process is largely unknown. Therefore, to address the mechanism of cancer inflammation we investigated the effects of an oncogene in a murine model system. A comprehensive genetic analysis revealed several soluble factors that were induced by RET/papillary thyroid carcinoma (PTC)3 gene expression including several proinflammatory cytokines, chemokines and immunologically relevant costimulatory molecules. Following a large genetic screen using RP3-expressing thyroid cells, we identified a highly abundant transcript and later identified it as interleukin 24 (Il24), a cytokine with diverse tumor suppressor and inflammatory activities. We show that RET/PTC3 induces Il24 expression in rat thyrocytes and that this expression is dependent on the signaling properties of its tyrosine kinase. Likewise, RET/PTC3 induces large amounts of Il24 following expression in murine thyrocytes, but its expression is dramatically reduced in poorly differentiated carcinomas, a finding that parallels the loss of RET/PTC3 expression. Consistent with its behavior as a tumor suppressor, the loss of Il24 coincided with the loss of RET/PTC3 in poorly differentiated mouse tumors. A functional role of Il24 in the autocrine growth/survival of RET/PTC3-expressing thyroid cells was identified helping to support its role in cellular transformation. These data suggest that the induction of Il24 by oncogenes may support tumor growth at the early stages of cancer.

RET/PTC3 (RP3) is an oncogenic fusion protein which is frequently expressed in papillary thyroid carcinomas and has been detected in thyroid tissue from patients diagnosed with Hashimoto's thyroiditis. The constitutive activation of the tyrosine kinase domain in the carboxyl-terminal end of RP3 induces signaling pathways within thyrocytes and causes cellular transformation. One of the signaling pathways activated in RP3-expressing cells involves the activity of the transcription factor NF-kappaB and the production of downstream targets including GM-CSF and macrophage chemotactic protein 1. These factors are known to be immunostimulatory, making RP3 a molecular adjuvant and potentially promoting tissue-specific immunity. However compelling, these in vitro data do not reliably predict gene function in vivo or the cumulative effects of time-dependent processes such as angiogenesis, inflammation, or the influence of genetic background. To address these issues, we analyzed the production of proinflammatory mediators in mouse thyroid organs and demonstrate consistency with in vitro studies performed previously that Il1alpha, Il1beta, Il6, and Tnfalpha and the enzyme Cox2 are produced by RP3-transgenic thyroid tissue, but absent from nontransgenic thyroids. Furthermore, we find that that the genetic background of the host is important in the observed RP3-induced inflammation and tumor progression. These findings provide support for the notion that oncogene-induced cytokine secretion is important for the development and progression of thyroid carcinomas in genetically permissive hosts.

Thyroid epithelial cells frequently express one or more members of the rearranged during transfection/papillary thyroid carcinoma (RET/PTC) fusion oncogene family during early stages of cancer, and fusion gene transcripts have been found in inflammatory conditions of the thyroid such as the autoimmune disease, Hashimoto's thyroiditis. Because these oncogenes encode chimeric proteins, novel RET/PTC epitopes may be targets of antitumor immune responses. We have been interested in the RET/PTC3 (RP3) fusion protein because this family member is more frequently expressed in radiation-induced and childhood papillary carcinomas than other members of the fusion oncogene family. We hypothesized that the activated kinase of c-RET, in the form of RP3, when expressed in patients with thyroid disease, presents an unusual altered self target for T cell recognition. Interestingly, we find that immunization with mouse RP3 protein can induce a strongly immunogenic response to RP3, although this response is not directed against the peptide comprising the unique fusion region. Rather, the responses are specific for the carboxyl-terminal portion of RP3 that is derived from the self protein c-RET. Furthermore, transplantation of RP3-expressing thyroid tumors into naive mice resulted in leukocytic infiltration, tumor rejection, and induction of RP3-specific T cells. Thus, the somatic fusion of two unrelated self proteins results in the development of a uniquely immunogenic response directed against self epitopes within RP3. These studies may better define the mechanisms controlling the initiation of thyroid-specific immune responses and provide insight into the design of novel molecules for invoking tumor-specific immunity.

We have isolated a Meis1a transgenic mouse line exhibiting recessive male-specific lethality and non-alcoholic fatty liver disease (NAFLD), which coincides with pubescence and is androgen-dependent. The phenotype is due to disruption of an endogenous locus, since other Meis1a transgenic lines do not exhibit these phenotypes. Necropsy analysis revealed hepatic microvesicular steatosis in pubescent male homozygous mice, which is absent in transgenic females. The transgene insertion site was localized to chromosome 1 and further refined by cloning the flanking regions. Sequence analysis shows that the integration site disrupts a putative metallo-beta-lactamase gene with a 21.3 kb deletion encompassing exons 5-7.

Objectives: Cyclooxygenases (COX) are enzymes that catalyze the conversion of arachidonic acid to prostaglandins. COX-2, unlike the constitutively expressed COX-1, is an inducible enzyme upregulated during cell proliferation and inflammation. More recently, COX-2 has been implicated in the development of numerous types of epithelial cancers. In addition, COX-2 is highly expressed in several inflammatory diseases. Because of its dual role in inflammation and cancer, we were interested in determining if COX-2 plays a role in the development of human thyroid carcinoma and Hashimoto's thyroiditis, an autoimmune condition frequently associated with thyroid malignancy., Materials and Methods: Twenty paraffin-embedded human tissue specimens, including normal, inflammatory, and neoplastic thyroid sections, were analyzed by immunohistochemical staining for expression of human COX-2. In addition, COX-2 protein expression was verified by Western blot in two specimens., Results: Immunohistochemical staining confirmed the presence of COX-2 in thyroid epithelial neoplasms, including papillary and follicular carcinomas. Moreover, COX-2 expression was observed in patients with Hashimoto's thyroiditis. COX-2 expression, however, was not observed in normal thyroid tissue, multinodular goiter, or anaplastic carcinoma., Conclusions: We have shown that cyclooxygenase-2 is expressed in thyroid carcinoma and thyroid epithelium from patients with Hashimoto's thyroiditis but not in normal thyroid. The expression of COX-2 in both of these thyroid pathologies may provide a basis for the relationship between carcinogenesis and autoimmunity.

To better understand the roles of NeuroD, a member of the basic helix-loop-helix transcription factor family, during the differentiation of olfactory receptor neurons, we studied the expression of NeuroD in developing and aging mouse olfactory epithelium (OE). During embryonic period, NeuroD expression is confined in the basal compartment of OE. During neonatal period, NeuroD expression is detected in the middle compartment and in the basal compartment of OE. In the adult, the number of NeuroD expressing cells in the basal compartment significantly decreased, while the NeuroD-positive cells in the middle compartment was maintained throughout lifetime. This dual phase expression pattern of NeuroD suggests multiple roles of NeuroD in the neurogenesis of ORNs.

Cancers develop and progress via activation of oncogenes and loss of tumor suppressor genes, a progression that can be recapitulated through cross breeding mouse strains harboring genetic mutations. To define the role of RET/PTC3, p53 and Fhit in thyroid carcinogenesis, we intercrossed RET/PTC3 transgenics with p53-/- mice. This new strain, RET/PTC3p53-/-, succumb to rapidly growing and strikingly large multilobed thyroid tumors containing mixtures of both well and poorly differentiated, highly proliferative follicular epithelial cells. Interestingly, transplanted tumors from RET/PTC3p53-/- mice grew in SCID but not syngeneic immunocompetent mice indicating that these advanced tumors were immunogenic. RET/PTC3 protein expression was reduced to undetectable levels in tumors of older mice suggesting that the continued elevated expression of RET/PTC3 may not be necessary for tumor progression. Similarly, expression of Fhit protein was reduced in early tumors and undetected in older tumors irrespective of tumor histopathology. In contrast to RET/PTC3p53-/- mice, RET/PTC3Fhit-/- mice did not develop advanced thyroid carcinomas. These studies support a model of human thyroid cancer whereby thyroid epithelium expresses RET/PTC3 protein at early stages of tumor development, followed by the reduction of RET/PTC3 and loss of p53 function with progressive reduction of Fhit protein expression coincident with malignant progression.

We have previously reported that the thyroid-targeted expression of the RET/PTC3 oncogene (Tg-RET/PTC3) in transgenic mice induces follicular hyperplasia with papillary architecture, resulting in a modest increase of the thyroid gland volume, followed by the appearance of papillary carcinomas in approximately 1-year-old animals. In order to analyze the genetic alterations that may cooperate with RET/PTC3 in the development or progression of thyroid tumors, we interbred Tg-RET/PTC3 mice with Tg-E7 transgenic mice, which express the E7 oncogene of the human papilloma virus 16 in thyroid cells. Tg-E7 mice develop large colloid goiters with small papillae and well-differentiated thyroid carcinomas in older animals. Here we show that thyroid lesions in Tg-RET/PTC3-Tg-E7 double transgenics were morphologically different from those occurring in Tg-RET/PTC3 mice, while they were virtually indistinguishable from those occurring in Tg-E7 mice. In addition, the coexpression of RET/PTC3 and E7 oncogenes neither enhanced the malignant phenotype nor reduced the latency period of thyroid lesions with respect to parental transgenic lines. We conclude that the coexpression of RET/PTC3 and E7 lacks any cooperative effect in the neoplastic transformation of thyroid cells and that the E7-induced thyroid phenotype is dominant with respect to the RET/PTC3 one.

Some investigators have found an increased incidence of papillary carcinoma (PC) of the thyroid in patients with Hashimoto's (autoimmune) thyroiditis (HT), which raises the possibility that there may be more than an incidental association between these 2 diseases. In this study, we analyzed the pathology of Hashimoto's-associated thyroid carcinomas to see if these tumors showed any distinctive features. The possible significance of solid cellular nodules as preneoplastic lesions in patients with HT was investigated. A review of all the cases of HT during a 16-year period yielded 30 PC and 3 follicular carcinomas (FC). Within the PC there were 7 (23%) follicular variants. Twenty (67%) of the PC showed various degree of intratumoral fibrosis, ranging from thick fibrous septa separating tumor nodules to almost complete obliteration of the tumor by the fibrosis, with only microscopic residual tumor nests. In most of the cases, the desmoplastic response within the tumors was of the fibromatosis-like type with dense hyalinized collagen and bland-appearing spindle cells. All the tumors, independently of the degree of fibrosis, showed the nuclear features of PC. No correlation was found between the degree of fibrosis in the tumors and the thyroid gland outside the tumors. There were tumors with marked fibrosis without fibrosis outside the tumors. Four cases of PC (13%) showed a growth pattern characterized by cystic spaces with thick hyalinized walls and focal papillary hyperplasia lined by flat and cuboidal epithelium, reminiscent of a vascular neoplasm. There were 4 atypical solid microscopic nodules with confluent cellularity; 2 of them associated with a PC and the other 2 with diffuse HT without PC. These nodules were composed of cells with clear nuclei and occasional grooves without nuclear pseudoinclusions. By immunohistochemistry, 2 of 3 nodules showed cytoplasmic reactivity for cytokeratin 19, and 2 of 3 nodules were positive for the RET/PTC (rearranged during transfection, papillary thyroid carcinoma) antibody. In summary, HT-associated PC may frequently display prominent stromal desmoplasia and a pseudovascular pattern, both of which can present diagnostic difficulties if the cytologic features of PC are not recognized because of the marked obliteration of the tumor by the fibrosis. Atypical nodules may represent a precursor lesion of PC in patients with HT.

Olfactory neuroblastoma (ONB) is a highly vascularized and malignant tumor arising in olfactory neuronal precursors from the paranasal sinuses. Previously, we showed that treatment of JFEN cells with transforming growth factor (TGF)-alpha caused them to differentiate and respond to chemical odorants, whereas basic fibroblast growth factor (bFGF) treated cells differentiated and died. In the present study we show that established ONB tumors treated with bFGF upregulate the bFGF receptor (FGFR1) prior to differentiation. This cellular differentiation was evidenced by bFGF-induced expression of the human runt homologue AML1 (PEBP2 alpha B, CBFA-2) that is highly expressed in developing olfactory neuroepithelium and TrkA, a preferred nerve growth factor receptor. Since TrkA is expressed in supporting cells, but not in mature olfactory neurons, we hypothesize that the expression of AML1 and TrkA in bFGF-treated JFEN cells induced supporting cell differentiation. Collectively, these results have implications for the treatment of patients afflicted with ONB., (Copyright 2000 Academic Press.)

Genetic analysis of human papillary thyroid carcinomas (PTC) has revealed unique chromosomal translocations that form oncogenic fusion proteins and promote thyroid tumorigenesis in up to 60% of tumors examined. Although, the majority of thyroid specific translocations involve the growth factor receptor c-RET, variant rearrangements of the receptor for nerve growth factor, NTRK1 have also been described. One such translocation, TRK-T1, forms a fusion protein composed of the carboxyl terminal tyrosine kinase domain of NTRK1 and the amino terminal portion of TPR (Translocated Promoter Region). To determine if TRK-T1 expression can cause thyroid cancer in vivo, we developed transgenic mice that express the human TRK-T1 fusion protein in the thyroid. Immunohistochemical analysis of TRK-T1 transgenic mouse thyroids revealed TRK-T1 staining within the thyroid follicular epithelium. In contrast to nontransgenic littermates, 54% of transgenic mice developed thyroid abnormalities that included follicular hyperplasia and papillary carcinoma. Furthermore, all transgenic mice examined greater than 7 months of age developed thyroid hyperplasia and/or carcinoma. These data support the conclusion that TRK-T1 is oncogenic in vivo and contributes to the neoplastic transformation of the thyroid.

The TCL1 gene, which is located on chromosome 14, plays a major role in human hematopoietic malignancies and encodes a 14-kDa protein whose function has not been determined. This gene is expressed in pre-B cells, in immature thymocytes, and, at low levels, in activated T cells but not in peripheral mature B cells and in normal cells. The Tcl1 protein is similar in its primary structure to a protein encoded by the mature T-cell proliferation gene (MTCP1). The MTCP1 gene is located on the X chromosome and has been shown to be involved in rare chromosomal translocations in T-cell proliferative diseases. The murine TCL1 gene resides on mouse chromosome 12 and is homologous to the human TCL1 and MTCP1 genes. Murine Tcl1 protein has 116 amino acid residues and shares 50% sequence identity with human Tcl1, while the human and mouse Mtcp1 are nearly identical, with conservative differences in only six residues. The TCL1 and MTCP1 genes appear to be members of a family of genes involved in lymphoid proliferation and T-cell malignancies. Our laboratory has undertaken the study of the Tcl1 and Mtcp1 proteins to determine the structure and the function of these related proteins. In the present report, we have produced, using a bacterial expression system, the purified murine Tcl1 protein and a mutant form of murine Tcl1 protein containing a cysteine to alanine mutation at amino acid position 85. The recombinant proteins were purified by chromatography on a Ni-NTA resin followed by reverse-phase FPLC using a buffer system at pH 7.9 and a polymer-based reverse-phase column. The murine Tcl1 recombinant protein displays limited solubility and forms disulfide-linked dimers and oligomers, while the mutant murine Tcl1 C86A protein has increased solubility and does not form higher order oligomers. The purified recombinant murine proteins were characterized by N-terminal sequence analysis, mass spectrometry, and circular dichroism spectroscopy. Initial results indicate that the mutant murine Tcl1 C86A protein is suitable for both NMR and X-ray crystallographic methods of structure determination., (Copyright 2000 Academic Press.)

TCL1 and TCL1b genes on human chromosome 14q23.1 are activated in T cell leukemias by translocations and inversions at 14q32.1, juxtaposing them to regulatory elements of T cell receptor genes. In this report we present the cloning, mapping, and expression analysis of the human and murine TCL1/Tcl1 locus. In addition to TCL1 and TCL1b, the human locus contains two additional genes, TCL1-neighboring genes (TNG) 1 and 2, encoding proteins of 141 and 110 aa, respectively. Both genes show no homology to any known genes, but their expression profiles are very similar to those of TCL1 and TCL1b. TNG1 and TNG2 also are activated in T cell leukemias with rearrangements at 14q32.1. To aid in the development of a mouse model we also have characterized the murine Tcl1 locus and found five genes homologous to human TCL1b. Tcl1b1-Tcl1b5 proteins range from 117 to 123 aa and are 65-80% similar, but they show only a 30-40% similarity to human TCL1b. All five mouse Tcl1b and murine Tcl1 mRNAs are abundant in mouse oocytes and two-cell embryos but rare in various adult tissues and lymphoid cell lines. These data suggest a similar or complementary function of these proteins in early embryogenesis.

Human olfactory neuroepithelium (OE) is situated within the olfactory cleft of the nasal cavity and has the characteristic property of continually regenerating neurons during the lifetime of the individual. This regenerative ability of OE provides a unique model for neuronal differentiation, but little is known about the structure and biology of human olfactory mucosa. Thus, to better understand neurogenesis in human OE, we studied the expression of olfactory marker protein (OMP), TrkB and NeuroD in human nasal biopsies and autopsy specimens and compared these data with those obtained from normal and regenerating mouse OE. We show that NeuroD and TrkB are coordinately expressed in human OE. Thus, by using these markers we have been able to extend the known boundaries of the human OE to include the inferior middle turbinate. In normal mouse OE, TrkB and OMP expression overlap in cells closest to the superficial layer, but TrkB is expressed more strongly in the lower region of this layer. In contrast, NeuroD expression is more basally restricted in a region just above the globose basal cells. These characteristic expression patterns of OMP, TrkB and NeuroD were also observed in the regenerating mouse OE induced by axotomy. These results support a role of NeuroD and brain-derived neurotrophic actor (BDNF), the preferred ligand for TrkB, in the maintenance of the olfactory neuroepithelium in humans and mice.

The ras family members and the tyrosine kinases RET and TRK are frequently activated in human tumors of the thyroid gland. To ascertain the effects of these oncogenes in cultured thyroid cells we have generated expression vectors containing activated versions of the three genes under the control of the thyroid-specific thyroglobulin gene promoter. Here we show that the expression of the three oncogenes differently affects thyroid differentiation. While the TRK-T1 oncogene interferes with the capability of thyroid cells of trapping iodide and only marginally affects thyroglobulin gene expression, both RET/PTC3 and N-ras(Gln61-Lys) induce a dramatic reduction of thyroglobulin mRNA and alleviate TSH dependency for cellular growth. However, none of the three oncogenes is able to induce the appearance of neoplastic transformation markers, such as growth in semisolid medium and tumorigenicity in athymic mice. This indicates that genetic events additional to TRK, RET, or N-ras activation are required for fully malignant transformation of thyroid cells.

Our research goal is to better understand the mechanisms controlling the initiation and progression of thyroid diseases. One such disease, papillary thyroid carcinoma (PTC), is the leading endocrine malignancy in the United States. Recently, a family of related fusion proteins, RET/PTC1-5, has been implicated in the early stages of PTC. Although all five members of this family have the c-RET proto-oncogene kinase domain in their COOH terminus, little is known about how these genes alter follicular cell biology. Consequently, to answer questions related to the mechanism of the RET/PTC fusion protein action, we have devised a molecular genetic strategy to study PTC using a mouse model of thyroid disease. A new member of this fusion oncogene family, RET/PTC3, which has been implicated in more cases of solid tumor carcinoma (79%) than PTC1 or PTC2 and predominates (80%) in radiation-induced thyroid cancer of children, was investigated in our study. We have generated transgenic mice expressing human RET/PTC3 exclusively in the thyroid. These mice develop thyroid hyperplasia, solid tumor variants of papillary carcinoma and metastatic cancer. This new transgenic line will be useful in deciphering the molecular and biological mechanisms that cause PTC and histological variations in humans.

Two related oncogenes, TCL-1 and MTCP-1, are overexpressed in T cell prolymphocytic leukemias as a result of chromosomal rearrangements that involve the translocation of one T cell receptor gene to either chromosome 14q32 or Xq28. The crystal structure of human recombinant MTCP-1 protein has been determined at 2.0 A resolution by using multiwavelength anomalous dispersion data from selenomethionine-enriched protein and refined to an R factor of 0.21. MTCP-1 folds into a compact eight-stranded beta barrel structure with a short helix between the fourth and fifth strands. The topology is unique. The structure of TCL-1 has been predicted by molecular modeling based on 40% amino acid sequence identity with MTCP-1. The identical residues are clustered inside the barrel and on the surface at one side of the barrel. The overall structure of MTCP-1 superficially resembles the structures of proteins in the lipocalin family and calycin superfamily. These proteins have diverse functions, including transport of retinol, fatty acids, chromophores, pheromones, synthesis of prostaglandin, immune modulation, and cell regulation. However, MTCP-1 differs in the topology of the beta strands. The structural similarity suggests that MTCP-1 and TCL-1 form a unique family of beta barrel proteins that is predicted to bind small hydrophobic ligands and function in cell regulation.

The TCL1 oncogene on human chromosome 14q32.1 is involved in the development of T cell leukemia in humans. These leukemias are classified either as T prolymphocytic leukemias, which occur very late in life, or as T chronic lymphocytic leukemias, which often arise in patients with ataxia telangiectasia (AT) at a young age. The TCL1 oncogene is activated in these leukemias by juxtaposition to the alpha or beta locus of the T cell receptor, caused by chromosomal translocations t(14:14)(q11:q32), t(7:14)(q35:q32), or by inversions inv(14)(q11:q32). To show that transcriptional alteration of TCL1 is causally involved in the generation of T cell neoplasia we have generated transgenic mice that carry the TCL1 gene under the transcriptional control of the p56(lck) promoter element. The lck-TCL1 transgenic mice developed mature T cell leukemias after a long latency period. Younger mice presented preleukemic T cell expansions expressing TCL1, and leukemias developed only at an older age. The phenotype of the murine leukemias is CD4-CD8+, in contrast to human leukemias, which are predominantly CD4+CD8-. These studies demonstrate that transcriptional activation of the TCL1 protooncogene can cause malignant transformation of T lymphocytes, indicating the role of TCL1 in the initiation of malignant transformation in T prolymphocytic leukemias and T chronic lymphocytic leukemias.

In human leukemias and lymphomas nonrandom chromosomal rearrangements cause changes in cell growth and/or survival in such a way as to promote malignancy. The detailed study of the biochemical and genetic pathways altered in human cancer requires the identification or development of models to allow the study and manipulation of cancer gene function. Recently, the breakpoint gene TCL1, involved in chromosome translocations observed mostly in mature T-cell proliferations and chronic lymphocytic leukemias (CLL), was isolated and characterized, and showed to be part of a new gene family of proteins involved in these tumors. The murine Tcl1 gene, is similar in sequence to the murine and human MTCP1 gene also involved in T cell leukemias. The murine Tcl1 gene was shown to reside on mouse chromosome 12 in a region syntenic to human chromosome 14. Furthermore, we show that the murine Tcl1 gene is expressed early in mouse embryonic development and demonstrates expression in fetal hematopoietic organs as well as in immature T and B cells. Characterization of the murine Tcl1 gene will help in developing a mouse model of CLL and would provide the best opportunity to study and decipher the role of TCL1 in malignant transformation.

Hashimoto's thyroiditis is an inflammatory disease of the thyroid gland with autoimmune etiology. Patients afflicted with Hashimoto's have a higher risk of thyroid malignancies such as papillary thyroid carcinoma. In the present study, we investigated the frequency of papillary thyroid carcinoma specific genes in patients diagnosed with Hashimoto's disease. The newly identified oncogenes RET/PTC1 and RET/PTC3 provide useful and specific markers of the early stages of papillary carcinoma as they are highly specific for malignant cells. Using a sensitive and specific reverse transcriptase-polymerase chain reaction (RT-PCR) assay, we found messenger RNA (mRNA) expression for the RET/PTC1 and RET/PTC3 oncogenes in 95% of the Hashimoto's patients studied. All Hashimoto's patients presenting without histopathologic evidence of papillary thyroid cancer showed molecular genetic evidence of cancer. These data suggest that multiple, independent occult tumors exist in these patients at high frequency.

Olfactory neuroblastoma is a rare malignancy of the olfactory mucosa that may be derived from the olfactory epithelium. To characterize this tumor, we cultured olfactory neuroblastoma cells in the presence or absence of growth factors (transforming growth factor alpha and basic fibroblast growth factor) known to affect olfactory tissue and assessed their responsiveness to known odorants by measuring changes in intracellular calcium. Untreated cells did not respond to odorants. Basic fibroblast growth factor treatment had cytotoxic effects, and treated cells did not respond to odorants. Transforming growth factor alpha treatment resulted in the induction of odor responsiveness in these cells. Cells responded to odorants at 100 nM to 100 microM concentrations and responded with both increases and decreases in intracellular calcium. Increases in intracellular calcium were mediated by a calcium influx and were reversibly blocked by compounds known to inhibit second messenger pathways in olfactory receptor neurons. The calcium responses of the olfactory neuroblastoma cells were thus specific to the odorants and similar to those found in olfactory receptor neurons. The results support the notion that olfactory neuroblastoma cells may be of olfactory origin and thus they can be used as a model cell line to study human olfaction.

To study the molecular function of genes expressed during preimplantation development, we isolated a novel maternal transcript SSEC (Stage Specific Embryonic cDNA)-26 from a partial subtraction library of mouse unfertilized eggs and preimplantation embryos. The SSEC-26 transcript is abundant in the unfertilized egg and also actively transcribed from the newly formed zygotic genome. On the basis of its expression in eggs and embryos, this new mouse gene is named Mem (maternal-embryonic) 3. The genomic locus of Mem3 has been mapped to Chromosome (Chr) 8 near the D8Mit78 marker and the glutaryl CoA dehydrogenase (Gcdh) locus. The deduced amino acid sequence of MEM3 resembles that of the yeast VPS (Vacuolar Protein Sorting) 35 in two separate domains. A cDNA sequence of the potential human homolog of Mem3 has been assembled with partial clones from the EST database and assigned to human Chr 16.

Olfactory neuroblastoma (ONB) is a rare neuronal malignancy of the olfactory mucosal. Markers used in the diagnosis of ONB do not distinguish ONB from other neuronal tumors or tumors with neuroendocrine features thus making the diagnosis of ONB difficult. Using a modified RT-PCR technique, we show that the human homologue of the Drosophila achaete-scute gene HASH1 is expressed in 6 primary and one metastatic ONB specimens, whereas Olfactory Marker Protein (OMP) is not. Previous studies have shown that HASH1 is expressed in immature olfactory neurons and is required for their development. OMP, whose function is unknown, is expressed exclusively in mature olfactory neurons. Together, these data suggest that ONB is derived from immature olfactory neurons of neuroectodermal origin. Analysis of RNA expression in primary tumor specimens and in an established cell line make this an ideal system to study olfactory growth and differentiation. Furthermore, these studies represent the first molecular genetic analysis of this rare and unusual neuronal tumor.

Transcription of four members of the epidermal growth factor (EGF) family, EGF, TGF-alpha, Amphiregulin, and Cripto, was investigated in the ovulated unfertilized egg and preimplantation embryo using cDNA libraries. EGF was present as a maternal message only, TGF-alpha was present at low levels in each library, Amphiregulin was not detected, and Cripto transcripts were first detected in the blastocyst cDNA library. In situ hybridization studies of the implanting embryo revealed Cripto expression localized to the entire ectoderm and then to the rapidly growing ectoplacental cone. At gastrulation, Cripto was detected in the primitive streak and developing mesoderm. During organogenesis, Cripto localized to the developing heart. Two Cripto transcripts were detected: one is confined to the early embryo and teratocarcinoma cells, and the other, first found in the fetus, is the major form detected in adult organs.

Little is known about the repertoire of genes expressed following zygotic gene activation, which occurs during the two-cell stage in the mouse. As an initial attempt to isolate novel genes, we used previously prepared two-cell and two-cell subtraction cDNA libraries (Rothstein et al., Genes Dev 6:1190-1201, 1992) to isolate a panel of seven cDNA clones. Three cDNAs had no match in the current DNA sequence data banks and three others revealed sequence homology to portions of sequences in the data banks. One cDNA was 90% homologous to the ras-related gene Krev/rap 1A. The temporal patterns of expression of these genes during oocyte maturation and preimplantation development were analyzed by a reverse transcription-polymerase chain reaction (RT-PCR) assay developed to measure relative levels of mRNAs. Three distinct temporal patterns of expression, designated Classes 1-3, were found. The two Class 1 genes displayed an actin-like pattern, with a gradual decline in expression during oocyte maturation and through the two-cell stage, followed by increases at the eight-cell and/or blastocyst stages. The four genes in Class 2 were expressed at relatively high levels during oocyte maturation and through the one-cell stage and then declined abruptly between the one- and two-cell stages; an increase then occurred at the eight-cell and/or blastocyst stages. The expression of the gene in Class 3 declined during oocyte maturation, but then showed a transient increase at the one-cell stage, with only a very slight increase in synthesis at either the eight-cell or blastocyst stage.

The heterogeneity of the thymic stroma has made careful characterization of particular thymic stromal cell types difficult. To this end, we have derived a panel of cloned thymic stromal cell lines from simian virus 40 T antigen (SV40-T antigen) transgenic mice. Based on their analysis with monoclonal antibodies that distinguish among subsets of thymic stroma cells, and on the morphology and ultrastructural features of the different clones, we suggest that our panel includes representatives of the thymic subcapsular cortex or thymic nurse cells (427.1), the deep cortex or cortical reticular cells (1308.1) and the medulla including medullary interdigitating (IDC)-like cells (6.1.1) and medullary epithelial cells (6.1.7). A fifth cell type of undesignated but apparent medullary origin (6.1.11) was also isolated. All of the cell lines constitutively express the SV40 T antigen transgene and the class I antigens of the major histocompatibility complex (MHC), and they can be induced to express MHC class II antigens upon stimulation with recombinant interferon-gamma (IFN-gamma). These cell lines elaborate a factor(s) that induces the proliferation of cells from the fetal liver and bone marrow, but not from the neonatal thymus. A factor(s) elaborated by the 1308.1 cell line also induces the proliferation of fetal thymocytes in the absence of mitogens, phorbol esters or calcium ionophore which is augmented with the addition of recombinant interleukin-2 (IL-2). Analysis by reverse transcription polymerase chain reaction with primers for some mouse cytokines reveals that each of these cell lines contain granulocyte-macrophage colony-stimulating factor (GM-CSF) transcripts and that 1308.1, 6.1.1 and 6.1.7 produce IL-6 mRNA. Cell lines 1308.1 and 6.1.1 also produce IL-7; 6.1.1 produces IL-1 beta and tumor necrosis factor (TNF)-alpha while the 427.1 cell line produces IL-5 and IFN-gamma mRNA. None of the cell lines tested express the IL-2 receptor, IL-2, IL-3, IL-4, TNF-beta or macrophage inflammatory proteins mRNA. Conditioned medium (CM) from 1308.1 and 6.1.11 induced differentiation of cells purified from the mouse fetal liver into granulocytes; 1308.1 CM also induced differentiation of the mouse hematopoietic stem cell line 32DCl3(G) suggesting that the CM contains granulocyte (G)-CSF activity. Each cell line produces GM-CSF but the greatest activity is associated with 1308.1 and 6.1.11 CM. The availability of these well-characterized, functional, cloned thymic stromal cells will allow a more detailed analysis of the role of each cell type in both myeloid and T cell development.

By modifying current cDNA cloning and electroporation methods, large and representative murine cDNA libraries were synthesized from 10 to 100 ng mRNA isolated from unfertilized egg and preimplantation mouse embryos. High cloning efficiency is essential for complete representation of genes expressed in egg and preimplantation embryos and for the isolation of stage-specific genes using subtractive hybridization. Because the mouse embryo contains no more than 50 pg of poly(A)+ mRNA at any stage of preimplantation development, approximately 5000-10,000 embryos are required to obtain enough mRNA to synthesize libraries using current methods. To obtain a representative library that also includes rare transcripts, the size of the library should be at least 10(6) clones. The average percent conversion of mRNA to single-stranded cDNA was 20-40%, so that a cloning efficiency of nearly 2 x 10(8) cfu/microgram cDNA is required for such a cDNA library. No previous methods have provided directional cloning of cDNA into plasmids with these high efficiencies. The advent of electroporation methods for the introduction of nucleic acids into bacteria has made possible the use of standard plasmid vectors for high-efficiency cDNA cloning. Plasmid vectors are currently available that can accommodate the directional cloning of cDNA such that T7 and T3 RNA polymerase promoter sequences can be used to generate sense and anti-sense transcripts for subtractive hybridization and riboprobe synthesis. The cDNA libraries we derived using this methodology are a reusable and abundant source of genetic information about the control of preimplantation development. Specialized subtractive cDNA libraries enriched for genes expressed exclusively at a predetermined time in development give access to genes expressed in a stage-specific manner. The ability to construct new cDNA libraries from limited amounts of starting material ensures the provision of new and important resources for the identification and study of novel genes or gene families, and it is an important new tool for understanding the molecular control of mammalian development.

Substance abuse in the spinal cord injured (SCI) population has been addressed in a few controlled studies. These reports have led to the belief that many SCI patients were illicit drug users prior to their injury and that their drug abuse was a contributing cause of their accidents. One large study determined from answers obtained on a questionnaire that drug abuse, other than alcohol, was prevalent among injured veterans. Our study uses urine toxicology to determine the frequency of drug use and abuse, excluding alcohol, in 72 inpatients and 81 outpatients associated with an urban Veterans Affairs Medical Center. In a blinded experiment, urinary concentrations of opiates, barbiturates, amphetamines, methadone, benzodiazepines, and cocaine were determined with Syva reagents on an Abbott VP. Urinary cannabinoids were determined using Abbott TDX and the FPIA method. The use of illicit (unprescribed) drugs was surprisingly low (< or = 13 percent) for an urban medical center. Outpatient cannabinoid abuse was significantly more frequent than inpatient usage (p < 0.01). Barbiturates were not found in any patient. Benzodiazepines were taken most commonly (29 percent). Since benzodiazepine usage is the direct result of physician prescription, wide-spread usage of this agent is avoidable. Physicians should pay attention to the probable deleterious consequences of benzodiazepine addiction related to depressive effects on the central nervous system caused by its chronic use.