Postnatal neuronal apoptosis in the cerebral cortex: physiological and pathophysiological mechanisms.

In the first week of postnatal life of all examined mammalian species, there is a wave of apoptosis in the cerebral cortex, accounting for a loss of up to 30% of neuronal content from birth to adulthood. In this review we examine recent advances in the understanding of this curious phenomenon. We survey the phenomenological literature and elaborate a putative relationship between the formation of active neuronal networks and selective apoptosis of non-participatory neurons. The underlying reason for this apoptotic wave remains unclear, but molecular mechanisms are starting to be elucidated that account for its mechanism, including a role for insulin-like growth factor I (IGF-1) and the Rho GTPases RhoA and RhoB. In addition, we discuss pathophysiological situations in which a variety of common drugs used either recreationally or for medical purposes, or pharmacological blockade of N-methyl-d-aspartate receptor (NMDAR) function, can also cause massive levels of apoptosis in this same developmental window. Experimentation linking molecular causes of developmental and pathophysiological apoptosis in postnatal cerebral cortex is discussed.

Treatment of murine lupus with cDNA encoding IFN-gammaR/Fc.

IFN-gamma, a pleiotropic cytokine, is a key effector molecule in the pathogenesis of several autoimmune diseases, including lupus. Importantly, deletion of IFN-gamma or IFN-gammaR in several lupus-predisposed mouse strains resulted in significant disease reduction, suggesting the potential for therapeutic intervention. We evaluated whether intramuscular injections of plasmids with cDNA encoding IFN-gammaR/Fc can retard lupus development and progression in MRL-Fas(lpr) mice. Therapy significantly reduced serum levels of IFN-gamma, as well as disease manifestations (autoantibodies, lymphoid hyperplasia, glomerulonephritis, mortality), when treatment was initiated at the predisease stage, particularly when IFN-gammaR/Fc expression was enhanced by electroporation at the injection site. Remarkably, disease was arrested and even ameliorated when this treatment was initiated at an advanced stage. This therapy represents a rare example of disease reversal and makes application of this nonviral gene therapy in humans with lupus (and perhaps other autoimmune/inflammatory conditions) highly promising.

Elevated matrix metalloprotease and angiostatin levels in integrin alpha 1 knockout mice cause reduced tumor vascularization.

Integrin alpha1beta1 is a collagen receptor abundantly expressed on microvascular endothelial cells. As well as being the only collagen receptor able to activate the Ras/Shc/mitogen-activated protein kinase pathway promoting fibroblast cell proliferation, it also acts to inhibit collagen and metalloproteinase (MMP) synthesis. We have observed that in integrin alpha1-null mice synthesis of MMP7 and MMP9 was markedly increased compared with that of their wild-type counterparts. As MMP7 and MMP9 have been shown to generate angiostatin from circulating plasminogen, and angiostatin acts as a potent inhibitor of endothelial cell proliferation, we determined whether tumor vascularization was altered in the alpha1-null mice. Tumors implanted into alpha1-null mice showed markedly decreased vascularization, with a reduction in capillary number and size, which was accompanied by an increase in plasma levels of angiostatin due to the action of MMP7 and MMP9 on circulating plasminogen. In vitro analysis of alpha1-null endothelial cells revealed a marked reduction of their proliferation on both integrin alpha1-dependent (collagenous) and independent (noncollagenous) substrata. This reduction was prevented by culturing alpha1-null cells with plasma derived from plasminogen-null animals, thus omitting the source from which to generate angiostatin. Plasma from tumor-bearing alpha1-null animals uniquely inhibited endothelial cell growth, and this inhibition was relieved by the coaddition of either MMP inhibitors, or antibody to angiostatin. Integrin alpha1-deficient mice thus provide a genetically characterized model for enhanced angiostatin production and serve to reveal an unwanted potential side effect of MMP inhibition, increased tumor angiogenesis.

Expression of matrix metalloprotease-2-cleaved laminin-5 in breast remodeling stimulated by sex steroids.

The extracellular matrix plays an important role in breast remodeling. We have shown that matrix metalloprotease-2 (MMP2) cleaves laminin-5 (Ln-5), a basement membrane component, generating a fragment called gamma2x. Human breast epithelial cells, while constitutively immobile on intact Ln-5, acquire a motile phenotype on MMP2-cleaved Ln-5. We hypothesize that this mechanism may underlie cell mobilization across the basement membrane during branching morphogenesis in breast development regulated by sex steroids. We report that the expression of MMP2 and cleavage of Ln-5 correlate well with tissue remodeling and epithelial rearrangement of the breast both in vivo and in vitro. Thus, the Ln-5 gamma2x fragment was detected by immunoblotting in sexually mature, pregnant, and postweaning, but not in prepubertal or lactating mammary glands. Furthermore, cleaved Ln-5, as well as MMP2, became detectable in remodeling glands from sexually immature rats treated with sex steroids. In rat mammary gland explants, epithelial reorganization and luminal cell morphological changes were induced by the addition of exogenous MMP2, in parallel to the appearance of cleaved Ln-5. Similar effects were observed in epithelial monolayers plated on human Ln-5 and exposed to MMP2. These results suggest that cleavage of Ln-5 by MMP2 might be regulated by sex steroids and that it may contribute to breast remodeling under physiological and possibly pathological conditions.

Integrin signaling in fibrosis and scleroderma.

Integrins are a large family of heterodimeric transmembrane receptors for extracellular matrix proteins. As well as mediating cell attachment and the bulk of force transduction from the cytoskeleton, they convey signals from the extracellular matrix to the cell. alpha1beta1 and alpha2beta1 are the major collagen receptors in this family. a1beta1 provides negative feedback on collagen synthesis, whereas alpha2beta1 stimulates the synthesis of matrix metalloproteases. Each receptor modulates the signaling activity of the other to coordinate matrix synthesis and remodeling. Expression of both is reduced in scleroderma despite a paracrine environment which would be expected to upregulate them. Deficiencies in the integrins correlate with upregulated collagen synthesis and downregulated metalloprotease synthesis seen during the disease.

Mosaicism for a small marker chromosome resulting from a familial Robertsonian translocation (21;22).

A mosaic marker chromosome found in amniotic fluid was shown to have originated from the proximal part of the long arm of chromosome 22. This marker is unusual because it is the result of a deletion of a maternally inherited Robertsonian 21;22 translocation. It is suggested that the deletion and marker formation probably occurred post zygotically in the fetus. This rare case illustrates the difficulty in estimating risk of fetal abnormalities associated with de novo marker chromosomes. In this example, although the 'extra' marker chromosome contains euchromatin, the karyotype may still be 'balanced'.

Detection of mosaicism in amniotic fluid cultures: a CYTO2000 collaborative study.

PURPOSE: To evaluate the assumptions on which the American College of Medical Genetics (ACMG) Standards and Guidelines for detecting mosaicism in amniotic fluid cultures are based. METHODS: Data from 653 cases of amniotic fluid mosaicism were collected from 26 laboratories. A chi-square goodness-of-fit test was used to compare the observed number of mosaic cases with the expected number based on binomial distribution theory. RESULTS: Comparison of observed data from the in situ colony cases with the expected distribution of cases detected based on the binomial distribution did not reveal a significant difference (P = 0.525). CONCLUSIONS: The empirical data fit the binomial distribution. Therefore, binomial theory can be used as an initial discussion point for determining whether ACMG Standards and Guidelines are adequate for detecting mosaicism.

Evidence for a Turner syndrome locus or loci at Xp11.2-p22.1.

Turner syndrome is the complex human phenotype associated with complete or partial monosomy X. Principle features of Turner syndrome include short stature, ovarian failure, and a variety of other anatomic and physiological abnormalities, such as webbed neck, lymphedema, cardiovascular and renal anomalies, hypertension, and autoimmune thyroid disease. We studied 28 apparently nonmosaic subjects with partial deletions of Xp, in order to map loci responsible for various components of the Turner syndrome phenotype. Subjects were carefully evaluated for the presence or absence of Turner syndrome features, and their deletions were mapped by FISH with a panel of Xp markers. Using a statistical method to examine genotype/phenotype correlations, we mapped one or more Turner syndrome traits to a critical region in Xp11.2-p22.1. These traits included short stature, ovarian failure, high-arched palate, and autoimmune thyroid disease. The results are useful for genetic counseling of individuals with partial monosomy X. Study of additional subjects should refine the localization of Turner syndrome loci and provide a rational basis for exploration of candidate genes.

Integrin alpha1beta1 mediates a unique collagen-dependent proliferation pathway in vivo.

Activation of integrins upon binding to extracellular matrix proteins is believed to be a crucial step for the regulation of cell survival and proliferation. We have used integrin alpha1-null mice to investigate the role of this collagen receptor in the regulation of cell growth and survival in vivo. alpha1-deficient animals, which are viable and fertile, have a hypocellular dermis and a deficiency in dermal fibroblast proliferation as embryos. In vitro analysis of alpha1-null embryonic fibroblasts has revealed that their proliferation rate is markedly reduced when plated on collagenous substrata, despite normal attachment and spreading. Moreover, on the same collagenous matrices, alpha1-null fibroblasts fail to recruit and activate the adaptor protein Shc. The failure to activate Shc is accompanied by a downstream deficiency in recruitment of Grb2 and subsequent mitogen-activated protein kinase activation. Taken together with the growth deficiency observed on collagens, this finding indicates that the alpha1beta1 is the sole collagen receptor which can activate the Shc mediated growth pathway. Thus, integrin alpha1 has a unique role among the collagen receptors in regulating both in vivo and in vitro cell proliferation in collagenous matrices.

Neurodevelopment of children exposed in utero to antidepressant drugs.

BACKGROUND: Many women of reproductive age have depression, necessitating therapy with either a tricyclic antidepressant drug or a drug, such as fluoxetine, that inhibits the reuptake of serotonin. Whether these drugs affect fetal neurodevelopment is not known. METHODS: We studied the children of 80 mothers who had received a tricyclic antidepressant drug during pregnancy, 55 children whose mothers had received fluoxetine during pregnancy, and 84 children whose mothers had not been exposed during pregnancy to any agent known to affect the fetus adversely. The children's global IQ and language development were assessed between 16 and 86 months of postnatal age by age-appropriate Bayley Scales of Infant Development or the McCarthy Scales of Children's Abilities (for IQ) and the Reynell Developmental Language Scales. RESULTS: The mean (+/-SD) global IQ scores were 118+/-17 in the children of mothers who received a tricyclic antidepressant drug, 117+/-17 in those whose mothers received fluoxetine, and 115+/-14 in those in the control group. The language scores were similar in all three groups. The results were similar in children exposed to a tricyclic antidepressant drug or fluoxetine during the first trimester and those exposed throughout pregnancy. There were also no significant differences in temperament, mood, arousability, activity level, distractibility, or behavior problems in the three groups of children. CONCLUSIONS: In utero exposure to either tricyclic antidepressant drugs or fluoxetine does not affect global IQ, language development, or behavioral development in preschool children.

Specific reduction in osteopontin synthesis by antisense RNA inhibits the tumorigenicity of transformed Rat1 fibroblasts.

Osteopontin (OPN) is a secreted phosphoglycoprotein abundant in secretory luminal epithelia (Brown et al., 1992) and in bone (Reinholt et al., 1990). It contains a functional gly-arg-gly-asp-ser (GRGDS) integrin binding domain (Oldberg et al., 1986), promotes the adhesion of a variety of cell types (Somerman et al., 1989; Brown et al., 1992) and is a ligand for the vitronectin binding integrin alpha v beta 3 (Miyauchi et al., 1991). Elevated expression of OPN correlates with tumorigenic transformation in a great variety of stromal and epithelial cell lines (Senger et al., 1980, 1983, 1989; Craig et al., 1988; Chambers et al., 1992; Chang & Prince, 1993). The protein is also present in excess in the blood of patients with metastatic disease (Senger et al., 1988). To find whether OPN contributes significantly to the tumorigenic phenotype, we expressed antisense mRNA to OPN in high OPN producing malignant B77-Rat1 fibroblasts. This caused a reduction in their OPN secretion and reduced their ability to form both lung tumors in nude mice after intravenous injection, and colonies in soft agar. Antisense transfectants also showed increased spreading on vitronectin. These observations suggest that OPN overproduction is advantageous to the metastatic phenotype, possibly by altering adhesion via, or signal transduction from, vitronectin receptors.

Neurodevelopment of children exposed in utero to phenytoin and carbamazepine monotherapy.

OBJECTIVE: To compare pregnancy outcome prospectively after phenytoin and carbamazepine monotherapy with outcome in matched mother-child pairs exposed to nonteratogens to evaluate the relative fetal safety of these drugs. DESIGN: A prospective, controlled, and blinded observational study. PATIENTS: Thirty-six mother-child pairs exposed to carbamazepine monotherapy and 34 pairs exposed to phenytoin monotherapy, all prospectively studied, were compared with mother-child pairs exposed to nonteratogens. The controls were matched for maternal age, time of consultation, obstetric history, and socioeconomic status. MAIN OUTCOME MEASURE: The primary end point of interest was the children's global IQ measured by either the Bayley or the McCarthy scale according to their ages. SETTING: A teratology consultation program and two neurology services in Toronto, Ontario. RESULTS: Children exposed to phenytoin in utero had a mean (+/- SD) global IQ 10 points lower (95% confidence interval, 4.9 to 15.8 points) than their matched controls (113.4 +/- 13.1 and 103.1 +/- 25.1; P = .038). The Reynell language development scores followed a similar trend, with children exposed to phenytoin scoring significantly lower than their controls. Phenytoin-exposed children had a global IQ of 84 or less significantly more often than the control group (P < .01). Children exposed in utero to carbamazepine did not differ from their controls on any of the neurobehavioral tests. CONCLUSIONS: Our study suggests a clinically important negative effect of phenytoin on neurobehavioral development, independent of maternal or environmental factors, causing a substantial number of children to achieve a lower score than expected on cognitive tests. No similar effects could be shown after gestational use of carbamazepine.

Activin/inhibin beta B subunit gene disruption leads to defects in eyelid development and female reproduction.

Inhibins and activins are dimeric growth factors of the transforming growth factor-beta superfamily, a class of peptides that can regulate the growth and differentiation of a variety of cell types. Recently, activins have been implicated in early vertebrate development through their ability to evoke, in Xenopus embryo explants, both morphological and molecular changes characteristic of mesoderm induction. To understand these processes further, we have used homologous recombination in embryonic stem cells to create mouse strains carrying mutations in the gene encoding the activin/inhibin beta B subunit. These mice are expected to be deficient in activin B (beta B:beta B), activin AB (beta A:beta B), and inhibin B (alpha:beta B). Viable mutant animals were generated, indicating that the beta B subunit is not essential for mesoderm formation in the mouse. Mutant animals suffered, however, from distinct developmental and reproductive defects. An apparent failure of eyelid fusion during late embryonic development led to eye lesions in mutant animals. Whereas beta B-deficient males bred normally, mutant females manifested a profoundly impaired reproductive ability, characterized by perinatal lethality of their offspring. The phenotype of mutant mice suggests that activin beta B (1) plays a role in late fetal development and (2) is critical for female fecundity. In addition, we have found that expression of the related beta A subunit of activin is highly upregulated in ovaries of mutant females. Altered regulation of beta A activin in beta B-deficient mice may contribute to the mutant phenotype.

Predictive value of fine-needle aspiration of the thyroid in the classification of follicular lesions.

BACKGROUND: Fine-needle aspiration has been less valuable in the diagnosis of follicular lesions than for other neoplasms of the thyroid. It has been observed that follicular carcinoma is found in microfollicular, but not macrofollicular lesions, and this has served as a guide to management for many surgeons. The authors attempted to determine what cytologic parameters might usefully distinguish these types of follicular lesion. METHODS: The histologic findings and cytology of 56 thyroid lobectomies for follicular lesions that had adequate preoperative fine-needle aspiration of the thyroid (FNAT) were reviewed. Histologic specimens were classified into macrofollicular, mixed, and microfollicular groups. Cytologic features examined included the presence of colloid, irregularity of cell arrangements within groups, the presence and size of flat and folded cell sheets, three-dimensional clusters, and microfollicles and macrofollicles. RESULTS: Of the histologic specimens, 15 were preponderantly (> 70%) microfollicular, 19 were mixed, and 22 were predominantly (> 70%) macrofollicular. Three cytologic features were useful in their distinction: none of the predominantly microfollicular but 24% of the mixed and predominantly macrofollicular had abundant colloid (P = 0.048). Likewise, none of the predominantly microfollicular but 27% of the mixed and predominantly macrofollicular had large follicles (P = 0.026). Finally, 73% of the predominantly microfollicular but only 37% of the mixed and predominantly macrofollicular showed irregularity or crowding of cells in groups (P = 0.018). CONCLUSIONS: Used in combination, abundant colloid, regular spacing, and large follicles are helpful in distinguishing macrofollicular and mixed lesions from microfollicular ones, which have a higher malignant potential.

Glucocorticoid-dependent transformation by human papillomavirus type 16 E7 coding and 3' noncoding sequences.

The establishment of transformation of primary rodent cells by human papillomavirus (HPV) type 16 DNA requires glucocorticoid hormones (Pater et al., Nature 335, 832-835, 1988). Here we provide evidence by mutational analysis that, in the context of the hormone-regulated HPV 16 promoter/enhancer, the only protein coding sequences of HPV 16 required are those of the E7 gene. Moreover, additional sequences adjacent to the 3' end of E7 coding sequences are also essential for the establishment of the transformed phenotype. Splice donor sites, especially an E7 ORF 3' proximal one, are implicated for this cis-acting function, since specific deletion mutations of these splice sites greatly or completely reduced the frequency of transformation and the level of E7 RNA.

Choriocarcinoma following a partial hydatidiform mole: a case report.

Hydatidiform moles are classified as partial or complete by histologic criteria (Am J Obstet Gynecol 131:665-671, 1978 and Am J Obstet Gynecol 132:20-27, 1978). While persistent gestational trophoblastic tumors follow both types, there remains controversy as to whether the malignant extreme of gestational trophoblastic tumors, choriocarcinoma, can follow a partial hydatidiform mole (Am J Obstet Gynecol 127:167-170, 1977 and Arch Gynecol 234:161-166, 1984). In this instance, a 37-year-old woman presented with a partial hydatidiform mole that persisted and was treated with one course of chemotherapy. She attained a remission for 10 months, when a routine follow-up examination revealed an asymptomatic rise in serum beta-human chorionic gonadotropin from baseline to 14,600 mIU/mL. Dilatation and curettage revealed abundant avillous cytotrophoblast and syncytiotrophoblast with marked atypia, diagnostic of choriocarcinoma. Flow cytometry of paraffin blocks of both specimens showed the partial hydatidiform mole to be triploid and the choriocarcinoma diploid. The patient had no evidence of metastatic disease and was successfully treated with multiple-agent chemotherapy.

Granulomatous amebic encephalitis in an AIDS patient.

A 39-year-old man with AIDS died after developing a variety of neurologic symptoms and signs. CT showed multiple enhancing lesions in the cerebral hemispheres and cerebellum. Postmortem examination revealed parenchymal hemorrhagic and necrotizing lesions with a thrombo-occlusive vasculitis due to Acanthamoeba, which was typed as Acanthamoeba group 2, probably A rhysodes, by immunofluorescence.

The cloned butyrylcholinesterase (BCHE) gene maps to a single chromosome site, 3q26.

Human tissues have two distinct cholinesterase activities: acetylcholinesterase and butyrylcholinesterase. Acetylcholinesterase functions in the transmission of nerve impulses, whereas the physiological function of butyryl-cholinesterase remains unknown. An atypical form of butyrylcholinesterase or the absence of its activity leads to prolonged apnea following administration of the muscle relaxant suxamethonium. Inheritance of these butyrylcholinesterase variants is consistent with the enzyme activity being encoded in a single autosomal locus, BCHE (formerly CHE1 and E1), which has been assigned to chromosome 3. Previous in situ hybridization of a BCHE cDNA probe gave evidence of homologous sequences at 3q26 and 16q11-q23, raising the possibility of more than one locus coding for butyrylcholinesterase [H. Soreq, R. Zamir, D. Zevin-Sonkin, and H. Zakut (1987) Hum. Genet. 77: 325-328]. Using a different cDNA probe hybridized in situ to 46,XX,inv(3)(p25q21) metaphase chromosomes, we report here the localization of BCHE to a single autosomal location: 3q26.