O-GlcNAcylation regulates breast cancer metastasis via SIRT1 modulation of FOXM1 pathway.

Tumors utilize aerobic glycolysis to support growth and invasion. However, the molecular mechanisms that link metabolism with invasion are not well understood. The nutrient sensor O-linked-ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) modifies intracellular proteins with N-acetylglucosamine. Cancers display elevated O-GlcNAcylation and suppression of O-GlcNAcylation inhibits cancer invasion and metastasis. Here, we show that the regulation of cancer invasion by OGT is dependent on the NAD+-dependent deacetylase SIRT1. Reducing O-GlcNAcylation elevates SIRT1 levels and activity in an AMPK (AMP-activated protein kinase α)-dependent manner. Reduced O-GlcNAcylation in cancer cells leads to SIRT1-mediated proteasomal degradation of oncogenic transcription factor FOXM1 in an MEK/ERK-dependent manner. SIRT1 is critical for OGT-mediated regulation of FOXM1 ubiquitination and reducing SIRT1 activity reverses OGT-mediated regulation of FOXM1. Moreover, we show that SIRT1 levels are required for OGT-mediated regulation of invasion and metastasis in breast cancer cells. Thus, O-GlcNAcylation is a central component linking metabolism to invasion and metastasis via an SIRT1/ERK/FOXM1 axis.

Histopathological observations in the brains of children exposed to inhalational anesthetic agents: a retrospective autopsy-based study.

BACKGROUND: Numerous animal models have demonstrated neuronal damage resulting from anesthetic exposure in the developing brain. Studies have shown a relationship between anesthetic exposure and brain hypoxia, neurodegeneration and apoptosis. The relevance of data derived from controlled experimental studies to human neuropathology is a subject of debate. This study compares histopathological findings in post-mortem brain tissue specimens from children with and without exposure to inhalational anesthetic agents. METHODS: Autopsy reports were reviewed. Patients were divided into exposure and non-exposure groups defined as any procedure involving inhalational anesthetic agents. A retrospective chart review was performed collecting pathological findings of the brain. The autopsy results examined the presence of twelve different histopathological parameters reflecting morphologic changes in thirteen regions of interest in the central nervous system. RESULTS: Post-mortem neuropathological findings were analyzed. Thirteen different areas were focused upon and changes were categorized into twelve histopathological parameters. Gliosis, which was confirmed by immunohistochemical staining for glial fibrillary acidic protein, was more prevalent in the exposure group (N.=48) compared to the non-exposure group (N.=20) (P<0.05). CONCLUSION: The role of anesthetic neurotoxicity is not well understood. Numerous animal models have demonstrated neuronal apoptotic changes linked to anesthetic exposure, there is no tangible evidence supporting this relationship in humans. Our analysis demonstrates histopathological brain changes in children with anesthetic exposure not seen in the non-exposed group. Analysis was based on histopathological parameters representative of salient morphological findings of injury, which were encountered in anatomically divergent regions. Gliosis was the only statistically significant finding in post-mortem brain samples of patients who had received anesthetics.

[The role played by calcium in neuronal injury following neonatal hypoxia or convulsions]. / Función del calcio en la lesión neuronal tras hipoxia o convulsiones neonatales.

AIM: Calcium plays a complex and pivotal role both in neuronal development and function, and in hypoxia/ ischemia-induced cell death. In this paper, we review current concepts of calcium function emphasizing the neonatal period. DEVELOPMENT: Calcium enters the neuron through glutamate receptors (NMDA and AMPA) located on the neuronal membrane. After hypoxia or seizures, there is a conformational change of the receptors, with increased flow of calcium into the cytoplasm. Cytoplasmatic calcium triggers activation of several free-radical generation pathways, including the nitric oxide pathway, with a deleterious effect upon the neuron. Calcium then enters the neuronal nucleus, through specific receptors on the nuclear membrane. In our experience, hypoxia and neonatal seizures create nuclear membrane dysfunction, increasing the nitric-oxide-dependent flow of calcium into the nucleus. Nuclear calcium increase is critical for genetic transcription, pro-apoptotic gene activation and a cascade of biochemical and molecular events that lead to an increase of caspases and apoptotic neuronal death. CONCLUSIONS: Calcium has a crucial role in neuronal damage after neonatal hypoxia or seizures. A better knowledge of the pathogenic mechanisms that lead to neuronal damage after neonatal hypoxia or seizures will assist in future development of efficacious neuroprotective therapies.

[Cerebrovascular disorders in preterm neonates]. / Accidentes vasculares encefálicos en neonatos pretérmino.

AIM: To review the etiology, pathogenesis, clinical manifestations, diagnosis and treatment of cerebrovascular disorders (CVD) in preterm neonates. DEVELOPMENT: The germinal matrix-intraventricular hemorrhage (GM-IVH) is the most frequent intracranial hemorrhage. The pathogenesis includes intravascular, vascular and extravascular factors. The neuropathologic lesion is the hemorrhage within the subependymal GM. There are four degrees of severity. Clinical manifestations include three syndromes: catastrophic, saltatory and silent. Treatment is prophylactic. Periventricular hemorrhagic infarct is of venous origin, might be associated to GM-IVH and is difficult to differentiate from periventricular leukomalacia (PVL). Cerebellar hemorrhage frequency increases with decreasing gestational age. It may be asymptomatic or manifest with a non-specific neurologic picture. PVL is the most important ischemic lesion. Its pathogenesis includes anatomic, physiologic, maturational, biochemical, infectious and immunologic factors. The neuropathologic lesion is focal necrosis in the terminal distribution of the long penetrating arteries. Treatment is prophylactic. Other ischemic lesions are ischemic arterial infarct, cerebellar infarct, telencephalic leukoencephalopathy and pontosubicular necrosis. CVD diagnostic tests include cerebral ultrasound, computed tomography, magnetic resonance imaging and spectroscopy and electroencephalogram. Mortality of some CVD is high and some others are associated with a high risk of cerebral palsy and cognitive deficits. CONCLUSION: CVD are common in preterm neonates and represent an important cause of neurologic disease in the child.

Cerebellar hypoplasia with heterotopic purkinje cells in the molecular layer and preservation of the granule layers associated with severe encephalopathy. A new entity?

We present the case of an infant girl, born to first cousins, with a clinical phenotype consisting of microcephaly, hypotonia, strabismus and severe psychomotor retardation. Magnetic resonance imaging (MRI) showed global cerebellar atrophy involving the vermis and both hemispheres. The patient's serum transferrin levels were consistently unremarkable. Cerebellar biopsy, performed at 13 months of age, revealed heterotopic Purkinje cells in the molecular layer, but preservation of the external and internal granular layers. To our knowledge, this histological pattern of cerebellar cortical disorganization has not been described previously. The consanguinity of the parents suggests an autosomal recessive inheritance.

Hypoxia-induced caspase-3 activation and DNA fragmentation in cortical neurons of newborn piglets: role of nitric oxide.

Hypoxia results in generation of nitric oxide (NO) free radicals, activation of caspase-3, and genomic DNA fragmentation. The present study tests the hypothesis that hypoxia-induced caspase-3 activation and DNA fragmentation are nitric oxide mediated. Studies were conducted in newborn piglets, divided into normoxic (n = 5), hypoxic (n = 5), and hypoxic-7-NINA (n = 6). Hypoxic-7-NINA group received the neuronal nitric oxide synthase inhibitor, 7-Nitroindazole (7-NINA). Caspase-3 activity was determined spectrofluorometrically using enzyme-specific substrates. Sections from the neocortex were stained with an antiserum recognizing active caspase-3. Purified DNA was separated by gel electrophoresis. Administration of 7-NINA resulted in decreased immunoreactivity of caspase-3 (mean LI: 20.2%) as compared to the untreated hypoxia group (mean LI: 57.5%) (P < 0.05). 7-NINA attenuated caspase-3 enzymatic activity as well in comparison to the untreated hypoxia group (P < 0.05). Furthermore, multiple low molecular weight bands corresponding to DNA fragments were present in the hypoxic but not in the normoxic or hypoxic-7-NINA groups. Inhibition of nNOS abates the hypoxia-induced increase in active caspase-3 immunoreactivity, as well as enzymatic activity in cortical neurons, and DNA fragmentation in brain homogenates. We conclude that the coordinate increase of capase-3 activity and fragmentation of nuclear DNA in the hypoxic newborn piglet brain are NO mediated.

[Cerebral palsy: new pathogenetic concepts]. / Parálisis cerebral: nuevos conceptos etiopatogénicos.

OBJECTIVE: To review the current knowledge pertaining to the new pathogenetic aspects of cerebral palsy (CP). DEVELOPMENT: CP is a group of static, heterogeneous clinical syndromes, characterized by abnormal postural mechanisms and motor activities. Its prevalence in industrialized countries is 2 2.5/1000 newborns. CP should be differentiated from certain genetic or metabolic conditions with which it can be mistaken. Some cases of CP have a genetic basis and they are inherited following a mendelian pattern or are determined by specific isolated genes. Recently, the elevation of certain coagulation factors (i.e., Leiden factor V mutation) and cytokines (i.e. interleukins, a tumor necrosis factor) and interferons have been related to CP pathogenesis. Hypocapnia with PaCO2< 35 mmHg represents a risk for periventricular leukomalacia (PVL) in premature infants. PVL pathogenesis is complex and includes a series of mechanisms that interact among them: fetal/maternal infection, immuneinflammatory reaction, prematurity, intraventricular hemorrhage/iron, ischemia/reperfusion, free radical production, maturational sensitivity of oligodendrocytes, and glutamate effect. Neuroradiological and neuropathological data have demonstrated a cortical anatomical substrate for the intellectual deficits associated with PVL in premature infants. CONCLUSIONS: Current knowledge about CP pathogenesis opens the possibility of early diagnosis and development of new treatments, both therapeutic and preventive.

Presence of oligoclonal T cells in cerebrospinal fluid of a child with multiphasic disseminated encephalomyelitis following hepatitis A virus infection.

We have investigated the clonality of beta-chain T-cell receptor (TCR) transcripts from the cerebrospinal fluid (CSF) and peripheral blood from a 7-year old child who developed a multiphasic disseminated encephalomyelitis following an infection with hepatitis A virus. We amplified beta-chain TCR transcripts by nonpalindromic adaptor (NPA)-PCR-Vbeta-specific PCR. TCR transcripts from only five Vbeta families (Vbeta13, Vbeta3, Vbeta17, Vbeta8, and Vbeta20) were detected in CSF. The amplified products were combined, cloned, and sequenced. Sequence analysis revealed in the CSF substantial proportions of identical beta-chain of TCR transcripts, demonstrating oligoclonal populations of T cells. Seventeen of 35 (48%) transcripts were 100% identical, demonstrating a major Vbeta13.3 Dbeta2.1 Jbeta1.3 clonal expansion. Six of 35 (17%) transcripts were also 100% identical, revealing a second Vbeta13 clonal expansion (Vbeta13.1 Dbeta2.1 Jbeta1.2). Clonal expansions were also found within the Vbeta3 family (transcript Vbeta3.1 Dbeta2.1 Jbeta1.5 accounted for 5 of 35 transcripts [14%]) and within the Vbeta20 family (transcript Vbeta20.1 Dbeta1.1 Jbeta2.4 accounted for 3 of 35 transcripts [8%]). These results demonstrate the presence of T-cell oligoclonal expansions in the CSF of this patient following infection with hepatitis A virus. Analysis of the CDR3 motifs revealed that two of the clonally expanded T-cell clones exhibited substantial homology to myelin basic protein-reactive T-cell clones. In contrast, all Vbeta TCR families were expressed in peripheral blood lymphocytes. Oligoclonal expansions of T cells were not detected in the peripheral blood of this patient. It remains to be determined whether these clonally expanded T cells are specific for hepatitis A viral antigen(s) or host central nervous system antigen(s) and whether molecular mimicry between hepatitis A viral protein and a host protein is responsible for demyelinating disease in this patient.

Detection of JC virus DNA sequences and expression of the viral regulatory protein T-antigen in tumors of the central nervous system.

JC virus (JCV) is a neurotropic polyomavirus infecting greater than 70% of the human population worldwide during early childhood. Replication of JCV in brains of individuals with impaired immune systems results in the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Furthermore, JCV possesses an oncogenic potential and induces development of various neuroectodermal origin tumors including medulloblastomas and glioblastomas in experimental animals. The oncogenecity of JCV is attributed to the viral early gene product, T-antigen, which has the ability to associate with and functionally inactivate well-studied tumor suppressor proteins including p53 and pRB: The observations from laboratory animal experiments have provided a rationale for examining the presence of the JCV DNA sequence and expression of the viral oncogenic protein in human brain tumors. We have examined 85 clinical specimens from the United Kingdom, Greece, and the United States, representing various human brain tumors including oligodendroglioma, astrocytoma, pilocytic astrocytoma, oligoastrocytoma, anaplastic astrocytoma, anaplastic oligodendroglioma, glioblastoma multiforme, gliomatosis cerebri, gliosarcoma, ependymoma, and subependymoma, for their possible association with JCV. We performed gene amplification techniques using a pair of primers that recognize the JCV DNA sequence, and we demonstrated the presence of the viral early sequence in 49 (69%) of 71 samples. More importantly, our results from immunohistochemistry analysis revealed expression of JCV T-antigen in the nuclei of tumor cells in 28 (32.9%) of 85 tested samples. These observations, along with earlier in vitro and in vivo data on the transforming ability of this human neurotropic virus invite additional studies to re-evaluate the role of JCV in the pathogenesis of human brain tumors.

Acute hypoxia-induced alterations of calbindin-D28k immunoreactivity in cerebellar Purkinje cells of the guinea pig fetus at term.

Purkinje cells (PCs) are vulnerable to hypoxic/ischemic insults and rich in calcium and calcium-buffering/sequestering systems, including calcium-binding proteins (CaBPs). Calbindin-D28k is an EF-hand CaBP, which is highly expressed in PCs where it acts primarily as a cellular Ca++ buffer. Elevation of [Ca++] in the cytosol and nuclei of PCs is pivotal in hypoxic/ischemic cell death. We hypothesize that hypoxia results in decreased concentration, or availability of calbindin-D28k in PCs, thereby decreasing their buffering capacity and resulting in increase of intracellular and intranuclear [Ca++]. Cerebellar tissues from normoxic fetuses were compared to fetuses obtained from term pregnant guinea pigs exposed to hypoxia [7% FiO2] for 60 min. The pregnant guinea pigs were either killed upon delivery immediately following hypoxia (Hx0h) or were subsequently allowed to recover for 24 h (Hx24h) or 72 h (Hx72h). Fetal brain hypoxia was documented biochemically by a decrease in brain tissue levels of ATP and phosphocreatine. Compared to normoxic fetuses, there is a predominantly somatodendritic loss or decrease of calbindin-D28k immunohistochemical staining in PCs of Hx0h (p < 0.005), Hx24h (p < 0.05), and Hx72h (p < 0.005) fetuses. Hypoxia-induced alterations of calbindin-D28k immunoreactivity are qualitatively similar at all time points and include a distinctive intranuclear localization in subpopulations of PCs. A similar trend is demonstrated by immunoblotting. Subpopulations of TUNEL+/calbindin-D28k- PCs lacking morphologic features of apoptosis or necrosis are demonstrated in Hx24h and Hx72h fetuses. The present study demonstrates an abrogating effect of perinatal hypoxia on calbindin-D28k immunoreactivity in cerebellar PCs. The perturbation of this Ca++ buffer protein in hypoxia-induced neuronal injury may herald delayed cell death or degeneration.

Aberrant localization of the neuronal class III beta-tubulin in astrocytomas.

BACKGROUND: The class III beta-tubulin isotype (betaIII) is widely regarded as a neuronal marker in development and neoplasia. In previous work, we have shown that the expression of betaIII in neuronal/neuroblastic tumors is differentiation dependent. In contrast, the aberrant localization of this isotype in certain nonneuronal neoplasms, such as epithelial neuroendocrine lung tumors, is associated with anaplastic potential. OBJECTIVE: To test the generality of this observation, we investigated the immunoreactivity profile of betaIII in astrocytomas. DESIGN: Sixty archival, surgically excised astrocytomas (8 pilocytic astrocytomas, WHO grade 1; 18 diffuse fibrillary astrocytomas, WHO grade 2; 4 anaplastic astrocytomas, WHO grade 3; and 30 glioblastomas, WHO grade 4), were studied by immunohistochemistry using anti-betaIII monoclonal (TuJ1) and polyclonal antibodies. A monoclonal antibody to Ki-67 nuclear antigen (NC-MM1) was used as a marker for cell proliferation. Antibodies to glial fibrillary acidic protein (GFAP) and BM89 synaptic vesicle antigen/synaptophysin were used as glial and neuronal markers, respectively. RESULTS: The betaIII immunoreactivity was significantly greater in high-grade astrocytomas (anaplastic astrocytomas and glioblastomas; median labeling index [MLI], 35%; interquartile range [IQR], 20%-47%) as compared with diffuse fibrillary astrocytomas (MLI, 4%; IQR, 0.2%-21%) (P <.0001) and was rarely detectable in pilocytic astrocytomas (MLI, 0%; IQR, 0%-0.5%) (P <.0001 vs high-grade astrocytomas; P <.01 vs diffuse fibrillary astrocytomas). A highly significant, grade-dependent relationship was observed between betaIII and Ki-67 labeling and malignancy, but this association was stronger for Ki-67 than for betaIII (betaIII, P <.006; Ki-67, P <.0001). There was co-localization of betaIII and GFAP in neoplastic astrocytes, but no BM89 synaptic vesicle antigen/synaptophysin staining was detected. CONCLUSIONS: In the context of astrocytic gliomas, betaIII immunoreactivity is associated with an ascending gradient of malignancy and thus may be a useful ancillary diagnostic marker. However, the significance of betaIII-positive phenotypes in diffuse fibrillary astrocytomas with respect to prognostic and predictive value requires further evaluation. Under certain neoplastic conditions, betaIII expression is not neuron specific, calling for a cautious interpretation of betaIII-positive phenotypes in brain tumors.

Differential expression of TGF-beta, IL-2, and other cytokines in the CNS of Theiler's murine encephalomyelitis virus-infected susceptible and resistant strains of mice.

Intracranial inoculation of susceptible SJL mice with Theiler's murine encephalomyelitis virus (TMEV) results in biphasic disease consisting of early acute disease, followed by late chronic demyelinating disease, associated with mononuclear infiltrates and demyelinating lesions. In contrast, resistant C57BL/6 (B6) mice develop only early acute disease. We employed cytokine-specific RT-PCR to determine the expression of cytokine transcripts in the CNS of TMEV-infected SJL and B6 mice. During early acute disease, we have found a strong proinflammatory (Th1) cytokine response in the CNS of both TMEV-infected SJL and B6 mice, demonstrated by the expression of transcripts for IFN-gamma, IL-1, IL-6, IL-12p40, and TNF-alpha. At 8 days postinfection (p.i.), TGF-beta1 and TNF-alpha transcripts were present at significantly higher levels (P < 0.01) in the CNS of SJL susceptible mice in comparison to those found in the CNS of B6 mice. Immunohistochemical staining revealed that TGF-beta protein was expressed in leptomeningeal mononuclear inflammatory cell infiltrates in the brain of SJL mice but not in B6 mice, at 8 days p.i. TGF-beta may be responsible for the failure of SJL mice to develop an effective anti-TMEV CTL response. During late chronic demyelinating disease, high levels of proinflammatory Th1 cytokines were found in the CNS of SJL mice, but not B6 mice. Significantly higher levels (P < 0.01) of anti-inflammatory cytokine transcripts (IL-4, IL-5, and IL-10 (Th2 cytokines) and TGF-beta) were found in the spinal cord of TMEV-infected SJL mice with chronic demyelinating disease than in the spinal cord of B6 mice during the same time period (39 or 60 days p.i.). These anti-inflammatory cytokines may contribute to the downregulation of the proinflammatory response in SJL mice. High levels of IL-2 transcripts and protein appeared transiently in the spinal cord of TMEV-infected SJL mice before the onset of demyelinating disease and coincided with an influx of new T cells into the CNS and/or expansion of remaining T cells that have not been eliminated after viral clearance.

Congenital macroglossal angiodysplasia ("Lymphangioendotheliomatosis").

A case of congenital lingual angiodysplasia with macroglossia in a 5-year-old girl is presented. A diffusely enlarged tongue was present at birth and continued to grow as the child aged. It was accompanied by impaired speech, difficulty in eating and breathing, and sleep apnea, necessitating surgical intervention. The fundamental lesion represents a complex vascular malformation of the lymphangioma-hemangioma type, involving extensively the deep musculature of the tongue. Multifocal and multicentric cavernous lymphangioma-like and hemangioma-like areas merge with benign angioendotheliomatous-like foci in a background of variable muscle degeneration and marked fibrosis. Neither a borderline nor an overtly malignant vasoformative neoplasm was present. Because of its distinctively widespread, multicentric intramuscular distribution, this lesion may be construed as a diffuse variant of lingual lymphangioma-hemangioma malformation, closely resembling a previously described case of macroglossal lymphangioendotheliomatosis.

Differential distribution of the neuron-associated class III beta-tubulin in neuroendocrine lung tumors.

OBJECTIVE: To study the immunoreactivity profile of the neuron-associated class III beta-tubulin isotype (beta III) in epithelial lung tumors. DESIGN: One hundred four formalin-fixed, paraffin-embedded primary and metastatic lung cancer specimens were immunostained with an anti-beta III mouse monoclonal antibody (TuJ1) and an anti-beta III affinity-purified rabbit antiserum. Paraffin sections from fetal, infantile, and adult nonneoplastic lung tissues were also examined. RESULTS: In the fetal airway epithelium, beta III staining is detected transiently in rare Kulchitsky-like cells from lung tissues corresponding to the pseudoglandular and canalicular but not the saccular or alveolar stages of development. beta III is absent in healthy, hyperplastic, metaplastic, and dysplastic airway epithelium of the adult lung. In contrast, beta III is highly expressed in small cell lung cancer, large cell neuroendocrine carcinoma, and in some non-small cell lung cancers, particularly adenocarcinomas. There is no correlation between expression of beta III and generic neuroendocrine markers, such as chromogranin A and/or synaptophysin, in pulmonary adenocarcinomas. Also, focal beta III staining is present in primary and metastatic adenocarcinomas (to the lung) originating in the colon, prostate, and ovary. beta III is expressed to a much lesser extent in atypical carcinoids and is rarely detectable in typical carcinoids and squamous cell carcinomas of the lung. The distribution of beta III in small cell lung cancer and adenocarcinoma metastases to regional lymph nodes and brain approaches 100% of tumor cells, which is substantially greater than in the primary tumors. CONCLUSIONS: In the context of neuroendocrine lung tumors, beta III immunoreactivity is a molecular signature of high-grade malignant neoplasms (small cell lung cancer and large cell neuroendocrine carcinoma) although its importance in atypical carcinoids must be evaluated further. In addition, beta III may be a useful diagnostic marker in distinguishing between small cell lung cancers and certain non-small cell lung cancers (poorly differentiated squamous cell carcinomas), especially in small biopsy specimens. To our knowledge, beta III is the only tumor biomarker that exhibits a substantially more widespread distribution in poorly differentiated than in better differentiated pulmonary neuroendocrine tumors. However, the significance of beta III phenotypes in non-small cell lung cancer, particularly adenocarcinoma, with respect to neuroendocrine differentiation and prognostic value, requires further evaluation.

Detection of human neurotropic JC virus DNA sequence and expression of the viral oncogenic protein in pediatric medulloblastomas.

Medulloblastoma represents greater than 25% of childhood intracranial neoplasms and is considered a highly malignant tumor. This tumor, which arises predominantly in the cerebellar vermis, preferentially affects children between the ages of 5 and 15. Although the etiology of medulloblastomas in humans remains unknown, results from several experiments have indicated that the human neurotropic JC virus (JCV) is able to induce cerebellar neoplasms in rodents that exhibit a phenotype similar to that of human medulloblastomas. JCV is a polyomavirus that is widespread in the human population, with infection occurring most frequently in early childhood. In this study, we have examined the possible association of JCV with human medulloblastomas. By using PCR techniques we demonstrate that 11 of 23 samples of tumor tissue contain DNA sequences corresponding to three different regions of the JCV genome. More importantly, we demonstrate the presence of DNA sequences encoding the N- and C-terminal regions of the JCV oncogenic protein, T antigen, in 11 of 23 samples and the production of T antigen in the nuclei of 4 samples of tumor tissue. These observations provide evidence for a possible association of JCV with human medulloblastomas.

Angiocentric CD3(+) T-cell infiltrates in human immunodeficiency virus type 1-associated central nervous system disease in children.

A significant proportion of brain tissue specimens from children with AIDS show evidence of vascular inflammation in the form of transmural and/or perivascular mononuclear-cell infiltrates at autopsy. Previous studies have shown that in contrast to inflammatory lesions observed in human immunodeficiency virus type 1 (HIV-1) encephalitis, in which monocytes/macrophages are the prevailing mononuclear cells, these infiltrates consist mostly of lymphocytes. Perivascular mononuclear-cell infiltrates were found in brain tissue specimens collected at autopsy from five of six children with AIDS and consisted of CD3(+) T cells and equal or greater proportions of CD68(+) monocytes/macrophages. Transmural (including endothelial) mononuclear-cell infiltrates were evident in one patient and comprised predominantly CD3(+) T cells and small or, in certain vessels, approximately equal proportions of CD68(+) monocytes/macrophages. There was a clear preponderance of CD3(+) CD8(+) T cells on the endothelial side of transmural infiltrates. In active lesions of transmural vasculitis, CD3(+) T-cell infiltrates exhibited a distinctive zonal distribution. The majority of CD3(+) cells were also CD8(+) and CD45RO+. Scattered perivascular monocytes/macrophages in foci of florid vasculitis were immunoreactive for the p24 core protein. In contrast to the perivascular space, the intervening brain neuropil was dominated by monocytes/macrophages, microglia, and reactive astrocytes, containing only scant CD3(+) CD8(+) cells. Five of six patients showed evidence of calcific vasculopathy, but only two exhibited HIV-1 encephalitis. One patient had multiple subacute cerebral and brainstem infarcts associated with a widespread, fulminant mononuclear-cell vasculitis. A second patient had an old brain infarct associated with fibrointimal thickening of large leptomeningeal vessels. These infiltrating CD3(+) T cells may be responsible for HIV-1-associated CNS vasculitis and vasculopathy and for endothelial-cell injury and the opening of the blood-brain barrier in children with AIDS.

Cellular distribution of retinoic acid receptor-alpha protein in serous adenocarcinomas of ovarian, tubal, and peritoneal origin: comparison with estrogen receptor status.

Retinoids are effective growth modulators of human ovarian carcinoma cell lines. Their effects are mediated by nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which are transcriptional factors and members of the steroid/thyroid receptor superfamily. To our knowledge, until now, the cellular distribution of RAR proteins in human ovarian tumor specimens is unknown. This study provides new data on the differential cellular localization of RAR alpha protein in 16 serous adenocarcinomas originating from the ovaries, fallopian tubes, and the peritoneum. Using an affinity-purified antiserum specific for RAR alpha and a monoclonal antibody recognizing the full-length estrogen receptor molecule (clone 6F11), we performed immunohistochemistry on frozen tissue sections and examined the relationship between RAR alpha and estrogen receptor protein expression by comparing the percentage of immunostained tumor cells for either receptor. Our findings indicate a strong linear relationship between the percentages of RAR alpha- and estrogen receptor-labeled tumor cells as determined by linear regression analysis (P < 0.005, r = 0.825). A modest inverse relationship was found between the percentage of RAR alpha-positive tumor cells and histological grade, attesting to a differentiation-dependent trend (P < 0.04). No significant relationship was found between RAR alpha-labeled cells and clinical stage (P = 0.139), site of tumor origin (ovaries versus fallopian tubes versus peritoneum) (P = 0.170), and primary versus metastatic lesion (P = 0.561). Thus, serous adenocarcinomas are capable of expressing RAR alpha and estrogen receptor despite high histological grade and advanced stage of neoplastic disease. Compared with the heterogeneous localization of RAR alpha in cancer cells, there was widespread RAR alpha immunoreactivity in tumor-infiltrating lymphocytes, vascular endothelial cells, and stromal fibroblasts, underscoring the value of immunohistochemistry in the accurate determination of RAR/(RXR) content in tumor specimens.

T cells and T-cell cytokine transcripts in the synovial membrane in patients with osteoarthritis.

The synovial membrane in osteoarthritis (OA) often exhibits inflammatory infiltrates, but the role of T cells in these infiltrates is not known. T-cell activation antigens were analyzed by immunohistochemistry, and T-cell cytokine transcripts were measured by competitive PCR in synovial membranes from patients with OA and rheumatoid arthritis (RA). Lymphoid cell aggregates, containing primarily CD3+ T lymphocytes, were found in 65% of patients with OA. Mononuclear cells expressing the activation antigens CD69, CD25, CD38, CD43, CD45RO, and HLA class II were present in both patient groups, although in higher numbers in patients with RA. Interleukin 2 (IL-2) transcripts were found in 10 of 18 patients with OA versus 12 of 13 patients with RA (P = 0.03). Gamma interferon (IFN-gamma) transcripts were detected in 9 of 18 patients with OA versus 10 of 13 patients with RA (not significant), whereas IL-10 transcripts were found in nearly all patients. IL-4 and IL-5 were not detected in any patients. The levels of IFN-gamma and IL-2 transcripts, normalized for T-cell number equivalents, were not statistically different between OA and RA, but the levels of IFN-gamma, normalized for total cell number equivalents, were lower in OA than in RA (P = 0.01). Synovial membranes that expressed IL-2 and IFN-gamma transcripts were more likely to have heavier infiltrations of T cells and cells bearing activation markers than synovial membranes that did not express these cytokines. The presence of activated T cells and TH1 cytokine transcripts in chronic joint lesions of patients with OA suggests that T cells contribute to chronic inflammation in a large proportion of these patients.