Targeted disruption of the GATA3 gene causes severe abnormalities in the nervous system and in fetal liver haematopoiesis.

GATA-3 is one member of a growing family of related transcription factors which share a strongly conserved expression pattern in all vertebrate organisms. In order to elucidate GATA-3 function using a direct genetic approach, we have disrupted the murine gene by homologous recombination in embryonic stem cells. Mice heterozygous for the GATA3 mutation are fertile and appear in all respects to be normal, whereas homozygous mutant embryos die between days 11 and 12 postcoitum (p.c.) and display massive internal bleeding, marked growth retardation, severe deformities of the brain and spinal cord, and gross aberrations in fetal liver haematopoiesis.

Selection of variable-joining region combinations in the alpha chain of the T cell receptor.

Most T lymphocytes express an antigen-specific receptor composed of two subunits, alpha and beta, each of which can exhibit structural variability. A complex selection process operates on T cells during development in the thymus such that cells expressing only particular alpha beta-receptors migrate to the periphery. The alpha-chain repertoire was dissected at different stages of the selection process by using the polymerase chain reaction (PCR) technique to amplify only those transcripts of a particular variable region gene (V58). Sequences from these V58 cDNAs reveal the predominant expression of four joining (J) segments by T cells in the adult thymus, suggesting that molecular or cellular processes select particular V alpha J alpha combinations during development. T cells expressing one of these V58J alpha chains appear to have been negatively selected at a later stage, since these transcripts were present in the spleen at approximately one-tenth the level in the thymus. Results also indicate that residues present at the V alpha J alpha junction may be important in an early selection process.

Gene expression analysis by transcript profiling coupled to a gene database query.

We describe an mRNA profiling technique for determining differential gene expression that utilizes, but does not require, prior knowledge of gene sequences. This method permits high-throughput reproducible detection of most expressed sequences with a sensitivity of greater than 1 part in 100,000. Gene identification by database query of a restriction endonuclease fingerprint, confirmed by competitive PCR using gene-specific oligonucleotides, facilitates gene discovery by minimizing isolation procedures. This process, called GeneCalling, was validated by analysis of the gene expression profiles of normal and hypertrophic rat hearts following in vivo pressure overload.

Sequence diversity of T cell receptor alpha chain transcripts from BALB/c thymus.

Most of the diversity in T cell receptor subunits resides in the region that is the equivalent of the CDR3 of immunoglobulins. In order to learn more about the relative contributions of the various mechanisms that generate this diversity we have analyzed the sequences of alpha chain transcripts from BALB/c thymus. The J alpha repertoire of BALB/c mice was examined by comparison of new J alpha sequences and previously published sequences. Among the 41 J alpha genes examined, most of the diversity is located at the 5' end, consistent with the notion that this region contacts the antigen. VJ junctional diversity was examined by sequencing various V alpha J alpha combinations derived from different stages of development. Deletion of bases from the ends of V and J genes does not occur with equal frequency. A greater number of bases were deleted on average from the ends of J genes. Bases were added at junctions frequently in isolates from adult animals, consistent with the presence of terminal deoxynucleotidyl transferase. However, there were short stretches of sequences at junctions which were also present at the 5' end of J genes. These findings extend recent observations that alpha chain genes use multiple mechanisms for generating diversity.

Analysis of T cell receptor transcripts using the polymerase chain reaction.

The immune system is composed of two major types of lymphocytes, called B and T cells, that recognize foreign antigens. Recognition of antigens is accomplished through the generation of a large repertoire of different cell surface receptors, called immunoglobulins (Igs) on B cells and T cell receptors (TCRs) on T cells. The elucidation of Ig structure and molecular genetics preceded that of the TCR because of the greater abundance of Ig protein and mRNA. Although studies of TCRs have recently shed light on many of the issues of T cell recognition, the process of examining TCR gene structure has been tedious. Such analyses are also difficult because of the time required for the production, maintenance, and culturing of T cell clones. This report describes several strategies that use the polymerase chain reaction (PCR) to analyze very rapidly the structure of TCRs. Specific manipulations of the amplified material are discussed, as are the advantages of using the PCR to study TCR diversity.

Recurrent panniculitis in a man with asthma receiving treatment with leukotriene-modifying agents.

Leukotriene-modifying drugs are novel agents introduced recently to treat asthma. Both 5-lipoxygenase inhibitors, such as zileuton, and leukotriene receptor antagonists, such as zafirlukast and montelukast, have proved effective in the treatment of asthma. To our knowledge, there have been no detailed reports regarding dermatologic manifestations of this class of drugs. This article describes an unusual case of erythema nodosum in a 46-year-old asthmatic man who received 2 different leukotriene modifiers.

Acquisition of oral phencyclidine self-administration in rhesus monkeys: effect of sex.

RATIONALE: There are increasing reports of sex differences in the etiology of drug abuse in humans. A nonhuman primate model is useful for examining sex as a variable in drug abuse. OBJECTIVES: To determine whether there are sex differences in the acquisition of oral phencyclidine (PCP) self-administration and to compare the effect of altered feeding conditions on drug self-administration in male and female monkeys. METHODS: Acquisition of orally delivered PCP was studied using 7 female and 11 male adult rhesus monkeys. Initially, the monkeys were not food restricted, and they were given access to water under concurrent fixed-ratio (FR) 1 schedules during daily 3-h sessions. Each lip-contact response on a drinking spout resulted in a 0.3 ml liquid delivery. After baseline levels of water intake were obtained for 5 days, water was replaced with PCP (0.125 mg/ml) at both drinking spouts. Body weights were then reduced to 85% of free-feeding weights, and the monkeys were fed 30 min before the session began. The FR value was increased from 1 to 2, 4, and 8, at both drinking spouts. As a final step in the procedure, water and PCP were concurrently available at the two spouts under FR 8 schedules. Acquisition of PCP-reinforced behavior was considered to have occurred if PCP intake was consistently greater than water intake. RESULTS: Lip-contact responses and liquid deliveries were not significantly different between the females and males throughout the acquisition period, but there was a significant increase in responding and decrease in liquid intake as FR increased, and a significant increase in PCP consumption due to food restriction that did not differ in males and females. On a milligram per kilogram basis, female monkeys consumed nearly twice as much PCP as the males; however, this effect was not significant. The females showed significantly higher PCP than water intake while the males consumed approximately equal amounts of PCP and water. Of the seven females, 100% met the acquisition criterion of significantly greater PCP than water intake, while only 36.4% of the males met the criterion. CONCLUSION: These results concur with previous rat studies and indicate that female monkeys are more likely than males to acquire drug-reinforced behavior.

Role of estrogen in the acquisition of intravenously self-administered cocaine in female rats.

Previous work from this laboratory has revealed that female rats acquired cocaine self-administration at a faster rate than male rats and that a greater percentage of females acquired self-administration [Psychopharmacology 144 (1999) 77.]. It has been suggested that sex differences in stimulant self-administration may be related to ovarian hormones, particularly estrogen. To investigate this possibility, we compared four groups (n = 10) of female rats: ovariectomized (OVX) treated with either estradiol benzoate (EB) or vehicle (VEH), and sham-operated intact (SH) females treated with either the antiestrogen tamoxifen (TAM) or VEH. An autoshaping procedure was used to train rats to lever press for intravenous infusions of cocaine (0.2 mg/kg). The criterion for cocaine acquisition was a mean of 100 self-administered infusions over five consecutive 6-h sessions. Results revealed that 70% of the OVX + EB group and 80% of the SH + VEH group acquired self-administration, while only 30% of the OVX + VEH group and 50% of the SH + TAM group met the acquisition criterion. Rats that had estrogen chemically or surgically blocked exhibited significantly less responding for cocaine over the acquisition testing period, and fewer of these rats met the acquisition criterion compared to intact rats and to OVX rats with estrogen (EB) replacement. The percentages for females with estrogen (70% and 80%) vs. those without (OVX, 30%) were similar to those reported for intact females (70%) and males (30%) in the previous study [Psychopharmacology (2000)]. Taken together, these results suggest that estrogen is a key factor influencing drug-seeking behavior in female rats, and it may underlie sex differences in drug-reinforced responding.

Important melanocytic lesions in childhood and adolescence.

Melanocytic nevi are common in children and adolescents, and the preponderance of these lesions are benign. Congenital melanocytic nevi, dysplastic nevi, and large numbers of common acquired nevi, however, may indicate an increased risk of malignant melanoma. With the exception, possibly, of giant congenital nevi, melanoma associated with these lesions generally occurs in adulthood. Nonetheless, some patients can be identified as being at increased risk for the development of melanoma during childhood. The poor prognosis associated with advanced melanoma and the curability of early lesions underscore the importance of prompt recognition of melanoma when it does occur in children. Furthermore, physicians who care for children are in a key position to decrease risk of melanoma throughout the lifespan by encouraging avoidance of excessive sun exposure during childhood.

Advances in Alzheimer's disease. A review for the family physician.

Alzheimer's disease accounts for approximately two thirds of all cases of dementia in the United States and $90 billion in health care costs annually. Clinical and laboratory diagnostic tools have been refined so that clinicians now can diagnose Alzheimer's disease with up to 90% accuracy. Criteria for clinical diagnosis have been outlined by a work group of the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association. Key diagnostic tools include a complete patient history, mental status testing, and a thorough diagnostic workup to exclude the possibility of a reversible disease mimicking the symptoms of Alzheimer's disease. Currently, management of Alzheimer's disease involves a two-pronged approach: behavioral-supportive care and pharmacologic control of disruptive behavioral symptoms. In the future, drug therapy may be available to maintain memory and cognitive function. Cholinesterase inhibitors, which block the decrease in choline acetyltransferase activity associated with Alzheimer's disease, appear promising. The realistic goal of health care providers at the present time, however, should be symptom control rather than disease reversal.

Prevalence of depressive symptoms in primary care.

BACKGROUND: Depression is one of the most common medical disorders seen in primary care practice. The purpose of this study was to estimate the prevalence of depressive symptoms in primary care patients across the United States, and to describe patient characteristics that may be associated with an increased likelihood of those symptoms. METHODS: Survey data were obtained from a sample of 75,858 patients who visited one of 765 participating primary care physicians for any reason from February 1991 to September 1991. The outcome measurement used was the index score for presence of depressive symptoms on the Zung Self-rating Depression Scale. RESULTS: The overall prevalence of clinically significant depressive symptoms was found to be 20.9%, but the percentage of patients citing depression as a reason for visit (1.2%) was markedly lower. Patients who perceived their health as poor were more likely to have severe depressive symptoms than patients who perceived their health as excellent. Women, those in older age groups, and those with lower levels of education were more likely to have clinically significant depressive symptoms than men, those in younger age groups, and those with higher levels of education. When classified by marital status within each sex, married men and women were the least likely to have clinically significant depressive symptoms. CONCLUSIONS: Clinically significant depressive symptoms are highly prevalent in primary care patients; however, depression is an infrequent patient complaint. There are certain patient characteristics that may cue the physician to consider depression in the differential diagnosis, particularly the patient's self-perception of his or her overall health status. In addition, certain other subsets of patients are at increased risk of depression, such as women, those in older age groups, and those of lower socioeconomic status.

Nonrandom use of J alpha gene segments. Influence of V alpha and J alpha gene location.

TCR genes (alpha, beta, gamma, and delta) undergo rearrangement during development in the thymus. The repertoire of alpha beta and gamma delta cells is shaped first by genetic processes that control rearrangement and expression and then by intercellular processes that "select" cells expressing only particular receptors. In this report, alpha chain transcripts from different stages of ontogeny were examined to determine the influence of V and J gene location on the thymic repertoire. J alpha gene segments were not used equally by V alpha genes. The frequency of J alpha use, and thus the alpha chain repertoire, was influenced by at least three factors: the location of the V gene, the location of the J gene, and the age of the animal. V genes that are proximal to the J alpha region preferentially use the most 5' J alpha gene segments. In contrast, a V alpha gene that is distal to the J alpha region was rarely joined to these same 5' J alpha gene segments in thymocytes from adult animals. We suggest that the data are most easily explained by the frequent occurrence of secondary rearrangements in which a V alpha J alpha rearranged gene would be replaced by the joining of a flanking V alpha gene to a flanking J alpha gene segment. Finally, the evidence suggests that there may be differences in the extent of secondary rearrangements between fetal and adult animals.

Characterization of transcripts from unrearranged V alpha 8 genes in the thymus.

The genes that encode the alpha- and beta-chains of the TCR undergo programmed rearrangement during differentiation of a T cell in the thymus, but it is not known what controls the order and specificity of the rearrangement event. By analogy with Ig genes, it is possible that transcription of an unrearranged V region gene may be necessary for "access" of the recombinase. To begin to address this issue, the six members of the V alpha 8 subfamily (five functional members and a pseudogene) from C57Bl/6 mice were examined. Consistent with the "accessibility" model, we show that unrearanged V alpha 8 genes are specifically expressed in the thymus of adult mice. Each of the functional genes was transcribed, but at different levels in the thymus. The five V alpha 8 genes were identical through the first 50 nucleotides of the 3' flanking region, and each contained an open reading frame that was contiguous with the V coding region. More interestingly, two of these putative proteins ended in Cys-X-Cys, a motif that is known to undergo isoprenoid modification. This finding and the conservation in the region that extends beyond the heptamer-nonamer region raise the possibility that some unrearranged V genes may encode proteins that have a novel function during early T cell development.

Mechanisms that generate junctional diversity in alpha and delta chains that use the Tcrd-V3 gene product.

The signals that dictate whether a thymocyte will express the alpha beta or gamma delta T-cell receptors are unknown. Although it is also not known if these two different cell types use identical recombinational machinery during rearrangement, the same variable (V) region genes can be used by both alpha and delta chains. By examining the products of rearrangements in alpha beta or gamma delta thymocytes that express identical V genes, we hoped to determine whether these cell types might differ in particular aspects of their recombinational activity. The polymerase chain reaction was used to show that the Tcrd-V2, Tcrd-V3, and Tcra-V3 genes are expressed as both Tcra and Tcrd transcripts in fetal and adult BALB/c mice. Sequencing of V delta 3 isolates was performed in order to compare the contribution of various mechanisms to the generation of junctional diversity. Extensive junctional diversity was present at all stages of development examined (fetal, newborn, and adult). During early development both alpha and delta chain junctional diversity is generated primarily by variability in the position of joining two gene segments (i.e., Tcrd-V3 to Tcra-J in alpha chains; Tcrd-V3 to Tcrd-D2 and Tcrd-D2 to Tcrd-J1 in delta chains). The pattern of base pair deletion from the end of the Tcrd-V3 gene was identical in alpha and delta chains and deletions occurred in fetal as well as adult T cells. In later development T cells use not only this mechanism for alpha and delta chains but also the addition of bases at gene segment junctions, presumably through the action of terminal deoxynucleotidyl transferase (TdT). Finally, a comparison of the variable domains of these alpha and delta chains shows that a notable difference is the variability in length of the CDR 3 region which can be significantly longer in delta-chains than in alpha-chains.

T-cell receptor delta-chain diversity in peripheral lymphocytes.

A small percentage (approximately 5%) of the cells in the adult thymus expresses a heterodimeric receptor, gamma delta, that exhibits extensive clonal diversity. The specificity and function of these cells are unclear. Furthermore, it is not known if their role in the immune system is primarily one that operates within the thymus during the selection of the T-cell repertoire or if they function primarily in an antigen-recognition capacity in the peripheral lymphoid system. To examine if gamma delta+ T cells in the periphery are as diverse as those in the thymus, we used the polymerase chain reaction to amplify delta-chain transcripts from polyclonal populations of thymic and splenic lymphocytes (the latter were derived from allogeneic mixed lymphocyte cultures). The nucleotide sequences of delta chains from the spleen, like those from the thymus, were all different. Most of the diversity was present in the region between the variable (V) and joining (J) gene segments and was generated through the use of the two known diversity (D) elements, D delta 1 and D delta 2, and by the addition or deletion of bases at the V delta D delta 1, D delta 1D delta 2, and D delta 2J delta junctions. The extensive gamma delta repertoire among peripheral cells suggests that they have the potential to recognize an array of ligands that could be as diverse as those recognized by alpha beta+ cells. The amplification strategy described here can be used to analyze rapidly the diversity exhibited by any of the members of the immunoglobulin-like gene families that undergo rearrangement.

Melanoma in children.

Four cases of malignant melanoma in children younger than 17 years of age are presented. Several preexisting conditions increase the risk of development of melanoma during childhood. These include giant congenital melanocytic nevi, the familial dysplastic nevus syndrome, and xeroderma pigmentosum. The role of small congenital lesions and sporadic dysplastic nevi in the development of melanoma in children is less clear. The signs and symptoms associated with melanoma in children are similar to those in adults, as are the histopathologic features, biologic behavior, and treatment of this tumor. The inadequacy of available therapy for metastatic melanoma underscores the necessity for the early diagnosis and prompt surgical treatment of melanomas in children.

Embryonic expression and cloning of the murine GATA-3 gene.

We describe the embryonic expression pattern as well as the cloning and initial transcriptional regulatory analysis of the murine (m) GATA-3 gene. In situ hybridization shows that mGATA-3 mRNA accumulation is temporally and spatially regulated during early development: although found most abundantly in the placenta prior to 10 days of embryogenesis, mGATA-3 expression becomes restricted to specific cells within the embryonic central nervous system (in the mesencephalon, diencephalon, pons and inner ear) later in gestation. GATA-3 also shows a restricted expression pattern in the peripheral nervous system, including terminally differentiating cells in the cranial and sympathetic ganglia. In addition to this distinct pattern in the nervous system, mGATA-3 is also expressed in the embryonic kidney and the thymic rudiment, and further analysis showed that it is expressed throughout T lymphocyte differentiation. To begin to investigate how this complex gene expression pattern is elicited, cloning and transcriptional regulatory analyses of the mGATA-3 gene were initiated. At least two regulatory elements (one positive and one negative) appear to be required for appropriate tissue-restricted regulation after transfection of mGATA-3-directed reporter genes into cells that naturally express GATA-3 (T lymphocytes and neuroblastoma cells). Furthermore, this same region of the locus confers developmentally appropriate expression in transgenic mice, but only in a subset of the tissues that naturally express the gene.

The histopathology of dysplastic nevi. Continued controversy.

The histopathologic criteria used in the diagnosis of dysplastic nevi have been a source of controversy, as has the clinical significance of these lesions. Several dermatopathologists noted for their work on dysplastic nevi were asked to contribute responses to questions regarding the architectural and cytological criteria used to classify a melanocytic nevus as dysplastic, the terminology used to describe these lesions, and the role of dysplastic nevi as precursors of melanoma. Although no consensus has been reached regarding the cytologic features required for diagnosis of dysplastic nevi, there is substantial agreement regarding the architectural features of these lesions.

GATA-2 and GATA-3 regulate trophoblast-specific gene expression in vivo.

We previously demonstrated that the zinc finger transcription factors GATA-2 and GATA-3 are expressed in trophoblast giant cells and that they regulate transcription from the mouse placental lactogen I gene promoter in a transfected trophoblast cell line. We present evidence here that both of these factors regulate transcription of the placental lactogen I gene, as well as the related proliferin gene, in trophoblast giant cells in vivo. Placentas lacking GATA-3 accumulate placental lactogen I and proliferin mRNAs to a level 50% below that reached in the wild-type placenta. Mutation of the GATA-2 gene had a similar effect on placental lactogen I expression, but led to a markedly greater reduction (5- to 6-fold) in proliferin gene expression. Placentas lacking GATA-2 secrete significantly less angiogenic activity than wild-type placentas as measured in an endothelial cell migration assay, consistent with a reduction in expression of the angiogenic hormone proliferin. Furthermore, within the same uterus the decidual tissue adjacent to mutant placentas displays markedly reduced neovascularization compared to the decidual tissue next to wild-type placentas. These results indicate that GATA-2 and GATA-3 are important in vivo regulators of trophoblast-specific gene expression and placental function, and reveal a difference in the effect of these two factors in regulating the synthesis of related placental hormones.