Pair housing, but not using a controlled reinforcer frequency procedure, attenuates the modulatory effect of probability presentation order on amphetamine-induced changes in risky choice.

Probability discounting is often measured with independent schedules. Independent schedules have several limitations, such as confounding preference for one alternative with frequency of reward presentation and generating ceiling/floor effects at certain probabilities. To address this potential caveat, a controlled reinforcer frequency schedule can be used, in which the manipulandum that leads to reinforcement is pseudo-randomly determined before each trial. This schedule ensures subjects receive equal presentations of the small and large magnitude reinforcers across each block of trials. A total of 24 pair-housed and 11 individually housed female Sprague Dawley rats were tested in a controlled reinforcer frequency procedure. For half of the rats, the odds against (OA) receiving the large magnitude reinforcer increased across the session (ascending schedule); the OA decreased across the session for half of the rats (descending schedule). Following training, rats received treatments of amphetamine (AMPH; 0, 0.25, 0.5, 1.0 mg/kg; s.c.). For pair-housed rats, AMPH (0.5 mg/kg) increased risky choice, regardless of probability presentation order, whereas a higher dose of AMPH (1.0 mg/kg) decreased discriminability of reinforcer magnitude for rats trained on the descending schedule only. For individually housed rats, probability presentation order modulated the effects of AMPH on probability discounting, as AMPH (0.25 and 0.5 mg/kg) increased risky choice in rats trained on the ascending schedule but not on the descending schedule. These results show that pair-housing animals, but not using a controlled reinforcer frequency procedure, attenuates the modulatory effects of probability presentation order on drug effects on risky choice.

Effects of d-amphetamine and MK-801 on impulsive choice: Modulation by schedule of reinforcement and delay length.

Procedural modifications can modulate drug effects in delay discounting, such as signaling the delay to reinforcement and altering the order in which delays are presented. Although the schedule of reinforcement can alter the rate at which animals discount a reinforcer, research has not determined if animals trained on different schedules of reinforcement are differentially affected by pharmacological manipulations. Similarly, research has not determined if using different delays to reinforcement can modulate drug effects in delay discounting. Male Sprague Dawley rats (n = 36) were split into four groups and were trained in a delay-discounting procedure. The schedule of reinforcement (fixed ratio [FR] 1 vs. FR 10) and delays to reinforcement (0, 5, 10, 20, and 50 s vs. 0, 10, 30, 60, 100 s) were manipulated for each group. Following behavioral training, rats were treated with d-amphetamine (0, 0.25, 0.5, and 1.0 mg/kg) and MK-801 (0, 0.03, and 0.06 mg/kg). Results showed that amphetamine decreased impulsive choice when a FR 1 schedule was used, but only when the short delay sequence was used. Conversely, amphetamine decreased impulsive choice when a FR 10 schedule was used, but only when rats were trained on the long delay sequence. MK-801 decreased impulsive choice in rats trained on a FR 1 schedule, regardless of delay sequence, but did not alter choice in rats trained on a FR 10 schedule. These results show that schedule of reinforcement and delay length can modulate drug effects in delay discounting.

Group I metabotropic glutamate receptor antagonists impair discriminability of reinforcer magnitude, but not risky choice, in a probability-discounting task.

The glutamatergic system has been identified as an important mediator of risky choice. However, previous studies have focused primarily on ionotropic glutamate receptors (e.g., NMDA receptors). Little research has examined the contribution of metabotropic glutamate receptors (mGluRs) on risky choice. The goal of the current experiment was to determine the effects of mGluR1 and mGluR5 antagonism on risky choice as assessed in probability discounting (PD). Male Sprague Dawley rats (n = 24) were trained in PD, in which consistently choosing a large, probabilistic reward (LR) reflects risky choice. For half of the rats, the odds against (OA) receiving the LR increased across blocks of trials, whereas the OA decreased across the session for half of the rats. Following training, rats received injections of the mGluR1 antagonist JNJ 16,259,685 (JNJ; 0, 0.1, 0.3, or 1.0 mg/kg; i.p) and the mGluR5 antagonist MTEP (0, 1.0, 3.0, or 10.0 mg/kg; i.p.). Regardless of which schedule was used, JNJ and MTEP decreased preference for the LR when its delivery was guaranteed. In contrast to delay discounting, in which blocking the mGluR1 has been shown to alter impulsive choice, these results show that the Group I mGluR family does not selectively alter risky choice. Instead, blocking these receptors appears to impair discriminability of reinforcers of varying magnitudes in PD.

Type 2 Gaucher disease with hydrops fetalis in an Ashkenazi Jewish family resulting from a novel recombinant allele and a rare splice junction mutation in the glucocerebrosidase locus.

Gaucher disease, the deficiency of the lysosomal enzyme glucocerebrosidase (EC 3.2.1.45), is frequently encountered in the Ashkenazi Jewish population. Carrier screening for Gaucher disease by enzyme analysis performed during a routine pregnancy indicated that both Ashkenazi parents were carriers. Screening for four common Gaucher mutations was subsequently performed on fetal and parental DNA. None of the common Ashkenazi mutations were identified. However, when exons 9-11 were amplified and digested with NciI to detect the L444P mutation, it appeared that the mother and the fetus had an unusual allele and that the expected paternal allele was not present. When the fetal amniocytes were found to have less than 2% of the normal glucocerebrosidase activity and a fetal sonogram revealed hydrops fetalis, the pregnancy was terminated. The diagnosis of severe type 2 Gaucher disease was confirmed at autopsy. Ultrastructural studies of epidermis from the fetus revealed the characteristic disruption of lamellar bilayers, diagnostic for type 2 Gaucher disease. In subsequent studies of the fetal DNA, long-template polymerase chain reaction amplification revealed one appropriately sized band (approximately 6.5 kb) and one smaller (approximately 5.2 kb) band. Sequencing of the approximately 5.2-kb fragment identified a novel fusion allele resulting from recombination between the glucocerebrosidase gene and its pseudogene beginning in intron 3. This fusion allele was inherited from the father. The result was confirmed by Southern blot analysis using the enzyme S8tII. Sequencing of the 6.5-kb fragment identified a previously described, although rare, T-to-G splice junction mutation in intron 10 of the maternal allele, which introduced an NciI site. The couple had a subsequent pregnancy which was also found to be affected. This case study identifies a novel recombinant allele and an unusual splice junction mutation, and demonstrates that even in the Ashkenazi population, screening for common mutations may not accurately identify the most severe forms of the disease.

Mosaic trisomy 16 ascertained through amniocentesis: evaluation of 11 new cases.

Trisomy 16, once thought to result uniformly in early pregnancy loss, has been detected in chorionic villus samples (CVS) from on-going pregnancies and was initially ascribed to a second, nonviable pregnancy. Prenatally detected trisomy 16 in CVS and its resolution to disomy has led to the reexamination of the viability of trisomy 16. This study evaluates 11 cases of mosaic trisomy 16 detected through second trimester amniocentesis. In 9 of the 11 cases, amniocenteses were performed in women under the age of 35 because of abnormal levels of maternal serum alpha-fetoprotein (MSAFP) or maternal serum human chorionic gonadotropin (MShCG). The other two amniocenteses were performed for advanced maternal age. Five of the 11 pregnancies resulted in liveborn infants, and six pregnancies were electively terminated. The liveborn infants all had some combination of intrauterine growth retardation (IUGR), congenital heart defects (CHD), or minor anomalies. Two of them died neonatally because of complications of severe congenital heart defects. The three surviving children have variable growth retardation, developmental delay, congenital anomalies, and/or minor anomalies. In the terminated pregnancies, the four fetuses evaluated by ultrasound or autopsy demonstrated various congenital anomalies and/or IUGR. Cytogenetic and fluorescent in situ hybridization studies identified true mosaicism in 5 of 10 cases examined, although the abnormal cell line was never seen in more than 1% of cultured lymphocytes. Placental mosaicism was seen in all placentas examined and was associated with IUGR in four of seven cases. Maternal uniparental disomy was identified in three cases. Mosaic trisomy 16 detected through amniocentesis is not a benign finding but associated with a high risk of abnormal outcome, most commonly IUGR, CHD, developmental delay, and minor anomalies. The various outcomes may reflect the diversity of mechanisms involved in the resolution of this abnormality. As 80% of these patients were ascertained because of the presence of abnormal levels of MSAFP or MShCG, the increased use of maternal serum screening should bring more such cases to clinical attention.

Sex chromosome markers: characterization using fluorescence in situ hybridization and review of the literature.

Fluorescence in situ hybridization (FISH) using biotin labeled X- and Y-chromosome DNA probes was utilized in the analysis of 23 sex chromosome-derived markers. Specimens were obtained through prenatal diagnosis, because of a presumptive diagnosis of Ullrich-Turner syndrome, mental retardation, and minor anomalies or ambiguous genitalia; three were spontaneous abortuses. Twelve markers were derived from the X chromosome and eleven from the Y chromosome; this demonstrates successfully the value and necessity of FISH utilizing DNA probes in the identification of sex chromosome markers. Both fresh and older slides, some of which had been previously G-banded, were used in these determinations. We have also reviewed the literature on sex chromosome markers identified using FISH.

Evidence for structural heterogeneity from molecular cytogenetic analysis of dicentric Robertsonian translocations.

Most Robertsonian translocations are dicentric, suggesting that the location of chromosomal breaks leading to their formation occur in the acrocentric short arm. Previous cytogenetic and molecular cytogenetic studies have shown that few Robertsonian translocations retain ribosomal genes or beta-satellite DNA. Breakpoints in satellite III DNA, specifically between two chromosome 14-specific subfamilies, pTRS-47 and pTRS-63, have been indicated for most of the dicentric 14q21q and 13q14q translocations that have been studied. We have analyzed the structure of 36 dicentric translocations, using several repetitive DNA probes that localize to the acrocentric short arm. The majority of the translocations retained satellite III DNA, while others proved variable in structure. Of 10 14q21q translocations analyzed, satellite III DNA was undetected in 1; 6 retained one satellite III DNA subfamily, pTRS-47; and 3 appeared to contain two 14-specific satellite III DNA sub-families, pTRS-47 and pTRS-63. In 10/11 translocations involving chromosome 15, the presence of satellite III DNA was observed. Our results show that various regions of the acrocentric short arm, and, particularly, satellite III DNA sequences, are involved in the formation of Robertsonian translocations.

Retrovirus-mediated transfer and expression of an allogeneic major histocompatibility complex class II DRB cDNA in swine bone marrow cultures.

The possibility of inducing transplantation tolerance by somatic gene transfer is under investigation in our miniature swine model. As a crucial step in this project, we have used a retroviral vector engineered to express both a drug-resistance gene (Neo) and a swine class II DRB cDNA to transduce porcine bone marrow (BM) cells. Analysis of cultured swine fibroblasts exposed to high-titer viral supernatants demonstrated that drug resistance had been conferred and that transferred vector sequences were transcribed appropriately. Similar transduction studies with swine BM demonstrated the transfer of drug resistance to as high as 14% of colony-forming unit-granulocyte-macrophage (CFU-GM). Using polymerase chain reaction (PCR) of cDNA, vector-derived allogeneic DRB transcripts were detected in colonies derived from primitive CFU-Mix and high proliferative potential-colony-forming cell (HPP-CFC), as well as in drug-resistant GM colonies grown from transduced bone marrow (BM) maintained in long-term BM cultures (LTBMCs) for up to 5 weeks. These results indicate that a significant proportion of both colony-forming progenitors and LTBMC-initiating cells were transduced with the DRB-recombinant retroviral vector and that both vector-derived genes were expressed in the differentiated progeny of these cells.

Prospects for human gene therapy.

Retroviral vectors containing marker genes and the sequence for human proteins have been used to transduce cultured lymphocytes, which have then been reinfused into patients. Circulating hematopoietic progenitor cells from human fetal cord blood obtained at the time of term and premature deliveries as early as 19 weeks of gestation have been shown to express such transduced genes in vitro. Cord blood cells from fetal sheep sampled and transduced ex vivo and transfused back in utero expressed marker genes up to two years after birth. Although the efficiency of gene transfer into cells and their long-term expression need to be improved, the potential exists for treating some genetic diseases after prenatal diagnosis either in utero or shortly after birth.

Retrovirus-mediated gene transfer into CD4+ and CD8+ human T cell subsets derived from tumor-infiltrating lymphocytes and peripheral blood mononuclear cells.

Studies were undertaken to test the susceptibility of individual T cell subpopulations to retroviral-mediated gene transduction. Gene transfer into human tumor-infiltrating lymphocytes (TIL) or peripheral blood mononuclear cells (PBMC) was carried out by transduction with an amphotropic murine retroviral vector (LNL6 or N2) containing the bacterial neoR gene. The presence of the neoR gene in the TIL population was demonstrated by Southern blot analysis, detection of the enzymatic activity of the gene product and by the ability of transduced TIL to proliferate in high concentrations of G418, a neomycin analog that is toxic to eukaryotic cells. The presence of the neoR gene in TIL did not alter their proliferation or interleukin-2 dependence compared to nontransduced TIL. The differential susceptibility of CD4+ and CD8+ lymphoid cells to the retro-virus-mediated gene transfer was then tested. Transduction of heterogeneous TIL cultures containing both CD4+ and CD8+ cells resulted in gene insertion into both T cell subsets with no preferential transduction frequency into either CD4+ or CD8+ cells. In other experiments highly purified CD4+ and CD8+ T cell subpopulations from either TIL or PBMC could be successfully transduced with the neoR gene as demonstrated by Southern blot analysis and detection of the gene product neophosphotransferase activity. No such activity of vector DNA could be detected in controls of nontransduced cells. In these highly purified cell subsets the distinctive T cell phenotypic markers were continually expressed after transduction, G418 selection and long-term growth. Clinical trials have begun in patients with advanced cancer using heterogeneous populations of CD4+ and CD8+ gene-modified TIL.

Gene transfer into humans--immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction.

BACKGROUND AND METHODS: Treatment with tumor-infiltrating lymphocytes (TIL) plus interleukin-2 can mediate the regression of metastatic melanoma in approximately half of patients. To optimize this treatment approach and define the in vivo distribution and survival of TIL, we used retroviral-mediated gene transduction to introduce the gene coding for resistance to neomycin into human TIL before their infusion into patients--thus using the new gene as a marker for the infused cells. RESULTS: Five patients received the gene-modified TIL. All the patients tolerated the treatment well, and no side effects due to the gene transduction were noted. The presence and expression of the neomycin-resistance gene were demonstrated in TIL from all the patients with Southern blot analysis and enzymatic assay for the neomycin phosphotransferase coded by the bacterial gene. Cells from four of the five patients grew successfully in high concentrations of G418, a neomycin analogue otherwise toxic to eukaryotic cells. With polymerase-chain-reaction analysis, gene-modified cells were consistently found in the circulation of all five patients for three weeks and for as long as two months in two patients. Cells were recovered from tumor deposits as much as 64 days after cell administration. The procedure was safe according to all criteria, including the absence of infections virus in TIL and in the patients. CONCLUSIONS: These studies demonstrate the feasibility and safety of using retroviral gene transduction for human gene therapy and have implications for the design of TIL with improved antitumor potency, as well as for the possible use of lymphocytes for the gene therapy of other diseases.

Prospects for gene therapy.

Experiments in animal models and human cells in vitro suggest that gene transfer using retroviral vectors may be useful to treat genetic diseases and to gain information that may improve treatment of other common diseases such as cancer. The approach to treatment of genetic diseases by inserting genes into bone marrow cells and experimental models, and a novel application of gene transfer technology to cancer research are discussed herein.

Retroviral vector-mediated transfer of the bacterial neomycin resistance gene into fetal and adult sheep and human hematopoietic progenitors in vitro.

We compared the efficiency of retroviral vector (N2)-mediated transfer of the bacterial neomycin resistance gene (NeoR) into adult and fetal hematopoietic progenitors of sheep and humans by assessing their ability to form colonies in the presence of lethal doses of the neomycin analogue G418 in vitro. Fetal cells from both sheep and humans exhibited a higher degree of NeoR transfer than adult cells. The overall level of NeoR expression was significantly higher for sheep than human cells. The transfer/expression of NeoR into adult human bone marrow hematopoietic progenitors was not affected by the presence or absence of T cells and monocyte/macrophages. The efficiency of NeoR transfer into both adult and fetal human cells, however, was improved when transduction was carried out in the presence of recombinant human interleukin-3 and granulocyte-macrophage colony-stimulating factor. These results demonstrate the greater efficiency of NeoR gene transfer into fetal hematopoietic progenitors, which may provide a basis for the relatively higher efficiency of the in utero approach to gene therapy.

Retroviral-mediated gene transfer into hemopoietic cells.

Retroviral vectors have provided a means for the introduction of functioning exogenous genes into the hematopoietic system of whole animals. Although these vectors are quite efficient in the mouse model, when applied to non-murine in vivo systems, the efficiency of gene transfer has diminished to impractical levels. Since in vivo analyses are expensive and time consuming, in vitro models have been developed to speed the evaluation of alternative protocols. Using in vitro colony assays, three approaches were evaluated for their ability to improve the infectivity of hematopoietic progenitor cells with retroviral vectors. Exogenously applied hematopoietic growth factors increased the proportion of hematopoietic colonies in vitro up to an average of 5 fold. When alternative sources of progenitors, such as fetal cord blood, were used, improvements in infection efficiency were also obtained. Finally, evidence was acquired suggesting that xenotropic packaging of vectors also improved infection efficiency.

Expression of human adenosine deaminase in nonhuman primates after retrovirus-mediated gene transfer.

Primate bone marrow cells were infected with a retroviral vector carrying the genes for human adenosine deaminase (h-ADA) and bacterial neomycin resistance (neor). The infected cells were infused back into the lethally irradiated donor animals. Several monkeys fully reconstituted and were shown to express the h-ADA and neor genes at low levels in their recirculating hematopoietic cells for short periods of time.

Gene therapy: efforts at developing large animal models for autologous bone marrow transplant and gene transfer with retroviral vectors.

Two new large animal models, non-human primates and fetal sheep, have been developed in an effort to determine the feasibility of using retroviruses for gene therapy. The retroviral vectors N2 and SAX have been used to introduce the genes for neomycin phosphotransferase (neoR, conferring resistance to the antibiotic G418) and human adenosine deaminase (ADA; EC 3.5.4.17), respectively. Varying levels of human ADA activity have been detected in six of the eight SAX-treated monkeys analysed. In the monkey with the greatest activity, human ADA levels approximately 0.5% of endogenous monkey ADA levels were detected. By in situ hybridization, roughly one in 100 bone marrow cells were found to express vector DNA. Sheep have been used for studies of the infectability of fetal blood progenitors in vivo. Blood cells were treated with the N2 vector at the 96th day of gestation, and marrow cells were assayed for the presence of G418-resistant haematopoietic progenitors, starting from one week after birth (62 days after treatment). Up to 33% of colony-forming progenitors were drug resistant initially and, although the proportion of resistant colony-forming units declined, a level of 10% has been found 153 days after transplantation. Human bone marrow has also been treated with the N2 vector, resulting in 1-2% G418-resistant progenitors.