Raj P. Kapur

Defects in sensory and autonomic ganglia and absence of locus coeruleus in mice deficient for the homeobox gene Phox2a

Phox2a is a vertebrate homeodomain protein expressed in subsets of differentiating neurons. Here, we show that it is essential for proper development of the locus coeruleus, a subset of sympathetic and parasympathetic ganglia and the VIIth, IXth, and Xth cranial sensory ganglia. In the sensory ganglia, we have identified two differentiation blocks in Phox2a-/- mice. First, the transient expression of dopamine-beta-hydroxylase in neuroblasts is abolished, providing evidence that Phox2a controls noradrenergic traits in vivo. Second, the expression of the GDNF receptor subunit Ret is dramatically reduced, and there is a massive increase in apoptosis of ganglion cells, which are known to depend on GDNF in vivo. Therefore, Phox2a appears to regulate conventional differentiation traits and the ability of neurons to respond to essential survival factors.

Mice with Mitochondrial Complex I Deficiency Develop a Fatal Encephalomyopathy

To study effects of mitochondrial complex I (CI, NADH:ubiquinone oxidoreductase) deficiency, we inactivated the Ndufs4 gene, which encodes an 18 kDa subunit of the 45-protein CI complex. Although small, Ndufs4 knockout (KO) mice appeared healthy until approximately 5 weeks of age, when ataxic signs began, progressing to death at approximately 7 weeks. KO mice manifested encephalomyopathy including a retarded growth rate, lethargy, loss of motor skill, blindness, and elevated serum lactate. CI activity in submitochondrial particles from KO mice was undetectable by spectrophotometric assays. However, CI-driven oxygen consumption by intact tissue was about half that of controls. Native gel electrophoresis revealed reduced levels of intact CI. These data suggest that CI fails to assemble properly or is unstable without NDUFS4. KO muscle has normal morphology but low NADH dehydrogenase activity and subsarcolemmal aggregates of mitochondria. Nonetheless, total oxygen consumption and muscle ATP and phosphocreatine concentrations measured in vivo were within normal parameters.

Gastrointestinal neuromuscular pathology: guidelines for histological techniques and reporting on behalf of the Gastro 2009 International Working Group

The term gastrointestinal neuromuscular disease describes a clinically heterogeneous group of disorders of children and adults in which symptoms are presumed or proven to arise as a result of neuromuscular, including interstitial cell of Cajal, dysfunction. Such disorders commonly have impaired motor activity, i.e. slowed or obstructed transit with radiological evidence of transient or persistent visceral dilatation. Whilst sensorimotor abnormalities have been demonstrated by a variety of methods in these conditions, standards for histopathological reporting remain relatively neglected. Significant differences in methodologies and expertise continue to confound the reliable delineation of normality and specificity of particular pathological changes for disease. Such issues require urgent clarification to standardize acquisition and handling of tissue specimens, interpretation of findings and make informed decisions on risk-benefit of full-thickness tissue biopsy of bowel or other diagnostic procedures. Such information will also allow increased certainty of diagnosis, facilitating factual discussion between patients and caregivers, as well as giving prognostic and therapeutic information. The following report, produced by an international working group, using established consensus methodology, presents proposed guidelines on histological techniques and reporting for adult and paediatric gastrointestinal neuromuscular pathology. The report addresses the main areas of histopathological practice as confronted by the pathologist, including suction rectal biopsy and full-thickness tissue obtained with diagnostic or therapeutic intent. For each, indications, safe acquisition of tissue, histological techniques, reporting and referral recommendations are presented.

Complex I deficiency due to loss of Ndufs4 in the brain results in progressive encephalopathy resembling Leigh syndrome

To explore the lethal, ataxic phenotype of complex I deficiency in Ndufs4 knockout (KO) mice, we inactivated Ndufs4 selectively in neurons and glia (NesKO mice). NesKO mice manifested the same symptoms as KO mice including retarded growth, loss of motor ability, breathing abnormalities, and death by ~7 wk. Progressive neuronal deterioration and gliosis in specific brain areas corresponded to behavioral changes as the disease advanced, with early involvement of the olfactory bulb, cerebellum, and vestibular nuclei. Neurons, particularly in these brain regions, had aberrant mitochondrial morphology. Activation of caspase 8, but not caspase 9, in affected brain regions implicate the initiation of the extrinsic apoptotic pathway. Limited caspase 3 activation and the predominance of ultrastructural features of necrotic cell death suggest a switch from apoptosis to necrosis in affected neurons. These data suggest that dysfunctional complex I in specific brain regions results in progressive glial activation that promotes neuronal death that ultimately results in mortality.

Critical Proinflammatory and Anti-Inflammatory Functions of Different Subsets of CD1d-Restricted Natural Killer T Cells during Trypanosoma cruzi Infection

ABSTRACT Trypanosoma cruzi infects 15 to 20 million people in Latin America and causes Chagas disease, a chronic inflammatory disease with fatal cardiac and gastrointestinal sequelae. How the immune response causes Chagas disease is not clear, but during the persistent infection both proinflammatory and anti-inflammatory responses are critical. Natural killer T (NKT) cells have been shown to regulate immune responses during infections and autoimmune diseases. We report here that during acute T. cruzi infection NKT-cell subsets provide distinct functions. CD1d−/− mice, which lack both invariant NKT (iNKT) cells and variant NKT (vNKT) cells, develop a mild phenotype displaying an increase in spleen and liver mononuclear cells, anti-T. cruzi antibody response, and muscle inflammation. In contrast, Jα18−/− mice, which lack iNKT cells but have vNKT cells, develop a robust phenotype involving prominent spleen, liver, and skeletal muscle inflammatory infiltrates comprised of NK, dendritic, B and T cells. The inflammatory cells display activation markers; produce more gamma interferon, tumor necrosis factor alpha, and nitric oxide; and show a diminished antibody response. Strikingly, most Jα18−/− mice die. Thus, in response to the same infection, vNKT cells appear to augment a robust proinflammatory response, whereas the iNKT cells dampen this response, possibly by regulating vNKT cells.

Androgenetic/biparental mosaicism in an infant with hepatic mesenchymal hamartoma and placental mesenchymal dysplasia.

Androgenetic/biparental mosaicism, in which a subset of cells has complete paternal uniparental disomy, is associated with placental mesenchymal dysplasia (PMD), which is compatible with fetal development, indicating that fetal organs could also have androgenetic/biparental mosaicism, but few cases of somatic mosaicism have been described. A hepatic mesenchymal hamartoma (HMH) was resected from an otherwise healthy, nondysmorphic, 11-month-old girl, whose prenatal development was complicated by PMD. Placenta, HMH, histologically normal liver, and other tissues were examined for androgenetic/biparental mosaicism by analysis of (1) polymorphic DNA microsatellite markers, (2) the methylation status of an imprinted gene, SNRPN, and (3) immunohistochemically detectable protein products of the imprinted genes p57KIP2 and PHLDA2. The patients liver, HMH, and 1 placental sample demonstrated an increased ratio of paternal to maternal alleles, indicating androgenetic/biparental mosaicism. The androgenetic component comprised 26% to 60% of the cells. Other tissues, including a 2nd placental sample, white blood cells, umbilical cord, and abdominal fascia, had no detectable androgenetic component. Methylation analysis confirmed a relative excess of the paternally imprinted SNRPN homolog in the normal liver, HMH, and placenta. Placental p57KIP2 immunoreactivity was consistent with androgenetic/biparental mosaicism, but neither p57KIP2 nor PHLDA2 immunohistochemistry were informative for HMH, because neither antigen was detected in control liver samples. We report androgenetic/biparental mosaicism in nonplacental tissues of an infant with PMD and provide the 1st description of genome-wide paternal uniparental disomy in HMH. Androgenetic/biparental mosaicism appears to play a role in the pathogenesis of HMH and other somatic lesions, particularly those associated with PMD.

A case of true hermaphroditism reveals an unusual mechanism of twinning

Traditionally twins are classified as dizygous or fraternal and monozygous or identical (Hall Twinning, 362, 2003 and 735–743). We report a rare case of 46,XX/46,XY twins: Twin A presented with ambiguous genitalia and Twin B was a phenotypically normal male. These twins demonstrate a third, previously unreported mechanism for twinning. The twins underwent initial investigation with 17-hydroxyprogesterone and testosterone levels, pelvic ultrasound and diagnostic laparoscopy. Cytogenetic analysis was performed on peripheral blood cells and skin fibroblasts. Histological examination and Fluorescence in situ hybridization studies on touch imprints were performed on gonadal biopsies. DNA analysis using more than 6,000 DNA markers was performed on skin fibroblast samples from the twins and on peripheral blood samples from both parents. Twin A was determined to be a true hermaphrodite and Twin B an apparently normal male. Both twins had a 46,XX/46,XY chromosome complement in peripheral lymphocytes, skin fibroblasts, and gonadal biopsies. The proportion of XX to XY cells varied between the twins and the tissues evaluated. Most significantly the twins shared 100% of maternal alleles and approximately 50% of paternal alleles in DNA analysis of skin fibroblasts. The twins are chimeric and share a single genetic contribution from their mother but have two genetic contributions from their father thus supporting the existence of a third, previously unreported type of twinning.

Dandy-Walker malformation complex: correlation between ultrasonographic diagnosis and postmortem neuropathology.

OBJECTIVE: This autopsy-based study was designed to evaluate sonographic and neuropathologic findings of fetuses diagnosed prenatally with Dandy-Walker malformation complex. METHODS: The retrospective study encompassed a series of 44 autopsy cases from 2 tertiary referral centers with a prenatal ultrasound diagnosis of Dandy-Walker malformation complex between 1995 and 2003. Ultrasound and pathology data from the cases and from age-matched controls were reviewed in a blinded manner. An unequivocal diagnosis of Dandy-Walker malformation complex from ultrasonography or pathology archival images required significant hypoplasia or aplasia of the cerebellar vermis. RESULTS: Neuropathologic examination failed to confirm the prenatal diagnosis of Dandy-Walker malformation complex in 59% (26/44, 95% confidence interval [CI] 44–72) of the cases. After standardized reevaluation of high quality archival sonograms and pathology images, concordance remained poor at 55% (6/11 cases, 95% CI 28–79). Sonographic features that favored concordance included marked enlargement of the cisterna magna (≥ 10 mm), complete aplasia of the vermis, and a trapezoid-shaped gap between the cerebellar hemispheres. This latter finding contrasted with a keyhole-shaped gap in fetuses with no cerebellar neuropathology. CONCLUSION: Correlation between a prenatal ultrasound diagnosis of Dandy-Walker malformation complex and autopsy neuropathology findings is poor. Unequivocal prenatal sonographic diagnosis of Dandy-Walker malformation complex should be reserved for cases with the classic findings of Dandy-Walker malformation, including enlargement of the cisterna magna, aplasia of the vermis, and a trapezoid-shaped, rather than keyhole-shaped, interhemispheric gap. LEVEL OF EVIDENCE: III

Cyclopamine-Induced Holoprosencephaly and Associated Craniofacial Malformations in the Golden Hamster: Anatomic and Molecular Events

ABSTRACT Holoprosencephaly is a complex congenital malformation of the brain and is often associated with a spectrum of facial anomalies ranging from normocephaly or nondiagnostic changes to cleft lip/palate (premaxillary dysgenesis), cebocephaly, ethmocephaly, and cyclopia. The primary insult is thought to occur during gastrulation, when prechordal mesenchyme and overlying anterior neural plate undergo complex developmental interactions. Exposure to cyclopamine, a steroid isolated from the desert plant Veratrum californicum, causes holoprosencephaly in mammalian embryos. We have begun to study the pathogenesis of cyclopamine-induced holoprosencephaly and associated craniofacial anomalies in Syrian golden hamsters (Mesocricetus auratus). Embryos were exposed to a single maternal dose of cyclopamine during gastrulation on embryonic day (E) 7.0. By E13.0, 62% of fetuses showed craniofacial malformations, including premaxillary dysgenesis, ocular hypotelorism, and cebocephaly. Facial anomalies were associated with absence of the premaxilla and abnormalities of the midline cranial base, particularly the ethmoid and sphenoid bones. Histological sections from cyclopamine-treated embryos at earlier stages showed marked deficiency of cranial mesenchyme derived from the rostral neural crest. Expression of two transcription factors, HNF-3β and Hox-b5, which have been implicated in specification of rostral and caudal neural crest cells, respectively, were examined immunohistochemically. Treatment with cyclopamine caused a transient loss of HNF-3β immunoreactivity in prechordal mesenchyme, but had no effect on Hox-b5 expression. The findings suggest that an early event in the pathogenesis of cyclopamine-induced holoprosencephaly may be altered expression of selected proteins in the prechordal mesenchyme and floor plate with secondary impaired development of the adjacent neural plate and cranial neural crest.

Choriodecidual Group B Streptococcal Inoculation Induces Fetal Lung Injury without Intra-Amniotic Infection and Preterm Labor in Macaca nemestrina

Background Early events leading to intrauterine infection and fetal lung injury remain poorly defined, but may hold the key to preventing neonatal and adult chronic lung disease. Our objective was to establish a nonhuman primate model of an early stage of chorioamnionitis in order to determine the time course and mechanisms of fetal lung injury in utero. Methodology/Principal Findings Ten chronically catheterized pregnant monkeys (Macaca nemestrina) at 118–125 days gestation (termu200a=u200a172 days) received one of two treatments: 1) choriodecidual and intra-amniotic saline (nu200a=u200a5), or 2) choriodecidual inoculation of Group B Streptococcus (GBS) 1×106 colony forming units (nu200a=u200a5). Cesarean section was performed regardless of labor 4 days after GBS or 7 days after saline infusion to collect fetal and placental tissues. Only two GBS animals developed early labor with no cervical change in the remaining animals. Despite uterine quiescence in most cases, blinded review found histopathological evidence of fetal lung injury in four GBS animals characterized by intra-alveolar neutrophils and interstitial thickening, which was absent in controls. Significant elevations of cytokines in amniotic fluid (TNF-α, IL-8, IL-1β, IL-6) and fetal plasma (IL-8) were detected in GBS animals and correlated with lung injury (p<0.05). Lung injury was not directly caused by GBS, because GBS was undetectable in amniotic fluid (∼10 samples tested/animal), maternal and fetal blood by culture and polymerase chain reaction. In only two cases was GBS cultured from the inoculation site in low numbers. Chorioamnionitis occurred in two GBS animals with lung injury, but two others with lung injury had normal placental histology. Conclusions/Significance A transient choriodecidual infection can induce cytokine production, which is associated with fetal lung injury without overt infection of amniotic fluid, chorioamnionitis or preterm labor. Fetal lung injury may, thus, occur silently without symptoms and before the onset of the fetal systemic inflammatory response syndrome.