Neurokinin-1 Receptor (NK1-R) Expression in the Brains of SIV-Infected Rhesus Macaques : Implications for Substance P in NK1-R Immune Cell Trafficking into the CNS

Recent studies suggest a link between neuropsychiatric disorders and HIV/SIV infection. Most evidence indicates that monocytes/macrophages are the primary cell type infected within the CNS and that they contribute to CNS inflammation and neurological disease. Substance P (SP), a pleotropic neuropeptide implicated in inflammation, depression, and immune modulation via interaction with its cognate receptor, the neurokinin 1 receptor (NK1-R), is produced by monocyte/macrophages. While the presence of NK1-R on neurons is well known, its role on cells of the immune system such as monocyte/macrophages is just beginning to emerge. Therefore, we have examined the expression of SP and NK1-R and their relationship to SIV/HIV encephalitis (SIVE/HIVE) lesions and SIV-infected cells. These studies demonstrated intense expression of SP and NK1-R in SIVE lesions, with macrophages being the principal cell expressing NK1-R. Interestingly, all of the SIV-infected macrophages expressed NK1-R. Additionally, we examined the functional role of SP as a proinflammatory mediator of monocyte activation and chemotaxis. These studies demonstrated that treatment of monocytes with SP elicited changes in cell-surface expression for CCR5 and NK1-R in a dose-dependent manner. Moreover, pretreatment with SP enhanced both SP- and CCL5-mediated chemotaxis. All of these findings suggest that SP and NK1-R are important in SIV infection of macrophages and the development of SIVE lesions.The neuropathogenesis of HIV infection is complex and has led to the development of nonhuman primate models using infection of macaques with the simian immunodeficiency virus (SIV). The human and simian immunodeficiency viruses are closely related and produce nearly identical conditions in their respective hosts, thus the rhesus macaque infected with SIV is the premier animal model for the study of AIDS pathogenesis in general and of the neuropathogenesis of AIDS in particular. Additionally, nonhuman primates are widely used in neuroscience research, including neurophysiologic and neurobehavioral studies. Recent studies suggest a link between psychiatric disorders like stress and depression and the neuropathogenesis of HIV and/or its progression to AIDS.^1,2 Neuroinvasion by SIV and HIV occurs early in infection at the time of peak viremia. The virus most likely enters the brain within cells of monocyte/macrophage lineage, and the perivascular macrophage is the primary cell type productively infected within the CNS.^3,4Approximately 25 to 30% of untreated HIV-infected adult humans develop a debilitating neurological disorder termed AIDS dementia complex (ADC).^5,6,7 A wide variety of cytokines, which include IL-1beta, IL-6, and TNF-α, chemokines (CCL2, CCL3, CCL4 and CCL5), adhesion molecules, and other molecules such as Substance P (SP) produced by different cell types have been implicated in this process.^1,4,8,9 Microscopic evaluation of brains from individuals with ADC reveals a broad spectrum of pathological features including neuronal changes, multifocal encephalitis, accumulation of inflammatory macrophages and multinucleated giant cells (MNGCs), cerebral cortical atrophy, and white matter pallor.^5,10 The histopathological substrate of ADC, referred to as HIV encephalitis (HIVE), is characterized by perivascular accumulation of macrophages and MNGCs in the CNS with abundant infection of brain macrophages.^4,11 In addition to the pathological manifestations within the brains of individuals with ADC, extensive neurobehavioral effects have been characterized and include impaired fine motor control and memory, altered emotional control, motor slowing, and possibly depression. Compelling data from recent studies reveal the significance of inflammatory mediators including the neuropeptide SP in the neuropathogenesis of AIDS^12,13,14 as well as in clinical neurobehavioral effects of depression.^2,3,4 Such findings suggest that SP participates in important aspects of immune-neural communication and likely contributes to immune modulation in HIV/SIV infection.SP is an 11-aa neuropeptide and is the most extensively studied and most abundant member of the tachykinin family. SP is synthesized mainly by primary sensory neurons, however recent studies have demonstrated that immune cells also express SP mRNA and protein during SIV/HIV-infection,^1,8 suggesting that it may contribute to neurological disease by acting on its preferred receptor, NK1-R, a G-protein–coupled receptor expressed on T-cells, B-cells, monocyte/macrophages, NK cells, astrocytes, and neurons.⁹ Activation of NK1-R by SP results in increased phagocytic response in macrophages, enhanced inflammatory cytokine production by immune cells, and possible induction of a chemotactic response in monocyte macrophages, thus facilitating immune cell trafficking at sites of inflammation or infection.^{16,17,18,19,20} SP has a role in AIDS, and results from recent in vivo studies^1,8,15,21,22 revealed that NK1-R antagonists have an antiviral effect,²³ likely through down-regulation of CCR5⁹ as well as immunomodulatory and antidepressive effects.^23,24 The major focus of existing work on SP (and its receptor-NK1-R) in macaques, however, has focused on its role as a neurotransmitter or neuromodulator and therefore the distribution of SP and NK1-R on neurons is well known, while little data exists on the in vivo distribution of SP and NK1-R on other cell types.Because the cellular expression of SP and its receptor NK1-R is not fully characterized in normal or SIV-infected macaques, we examined the cellular distribution/location and phenotype of SP and NK1-R expressing cells in the CNS in vitro and in vivo using multiple techniques and at various stages of infection in animals with or without SIVE. Such analysis allowed us to assess spatial correlations between the presence of SIV-infected cells and cells positive for SP and NK1-R. Additionally, we examined the functional aspects of SP/NK1-R signaling in monocyte activation and chemotaxis. Data from our immunofluorescence and in situ hybridization studies show that while NK1-R is expressed in astrocytes and neurons, it is intensely expressed in SIVE lesions. NK1-R expression was detected in all SIV-infected cells associated with SIVE lesions, of which macrophages were the predominant cell type and also the primary cell type expressing NK1-R. T-cells were found in small numbers in SIVE lesions and were rarely infected but were shown to express NK1-R when present. Our quantitative flow cytometric analysis demonstrates that SP functionally regulates NK1-R and CCR5 expression on macaque monocytes. Furthermore, pretreatment of monocytes with SP enhanced SP-mediated chemotaxis as well as CCL5-mediated chemotaxis. The enhancement of SP-mediated chemotaxis in the presence of CCL5 demonstrates cross talk between NK1-R and CCR5 signaling pathways. These findings suggest that SP contributes to SIV-associated neurological disease and suggest that SP also has a role in augmented cellular trafficking across the blood-brain barrier and thus the development of SIVE lesions. This insight into the function of SP and NK1-R allows for a better understanding of the interaction between the immune and nervous systems, and may lead to advancements in treatment of neurological and neuropsychiatric disease.     

A left ventricular epicardial to right ventricular endocardial dominant frequency gradient exists in human ventricular fibrillation

Purpose  

We hypothesized that in patients with left ventricular dysfunction undergoing implant of a biventricular ICD, the local dominant frequency during early induced ventricular fibrillation would be higher at an epicardial left ventricular position compared to an endocardial right ventricular position.     

Mutant mitochondrial elongation factor G1 and combined oxidative phosphorylation deficiency

Although most components of the mitochondrial translation apparatus are encoded by nuclear genes, all known molecular defects associated with impaired mitochondrial translation are due to mutations in mitochondrial DNA. We investigated two siblings with a severe defect in mitochondrial translation, reduced levels of oxidative phosphorylation complexes containing mitochondrial DNA (mtDNA)-encoded subunits, and progressive hepatoencephalopathy. We mapped the defective gene to a region on chromosome 3q containing elongation factor G1 (EFG1), which encodes a mitochondrial translation factor. Sequencing of EFG1 revealed a mutation affecting a conserved residue of the guanosine triphosphate (GTP)-binding domain. These results define a new class of gene defects underlying disorders of oxidative phosphorylation.     

The role of the complement system in innate immunity

Complement is a major component of innate immune system involved in defending against all the foreign pathogens through complement fragments that participate in opsonization, chemotaxis, and activation of leukocytes and through cytolysis by C5b-9 membrane attack complex. Bacterias and viruses have adapted in various ways to escape the complement activation, and they take advantage of the complement system by using the host complement receptors to infect various cells. Complement activation also participates in clearance of apoptotic cells and immune, complexes. Moreover at sublytic dose, C5b-9 was shown to promote cell survival. Recently it was also recognized that complement plays a key role in adaptive immunity by modulating and modifying the T cell responses. All these data suggest that complement activation constitutes a critical link between the innate and acquired immune responses.     

A novel mitochondria-targeting method using special staining for the detection of apoptotic hepatocytes

ABSTRACT

Early detection of apoptotic cells on histological slides is of major importance for both diagnostic and research areas. In the current study, the aim was to propose a convenient method to stain the mitochondria and establish whether hepatocytes undergoing apoptosis can be identified in tissue sections using the proposed method. Liver tissue from five adult chinchillas was fixed with 10% neutral buffered formalin for Goldner’s trichrome (GT) and Groat’s iron hematoxylin and eosin (HE) stains and with Kolster’s fixative for the Heidenhain’s iron hematoxylin procedure. The HE and GT-stained sections showed the morphological features consistent with apoptosis i.e., homogenous intensely acidophilic cytoplasm, cell shrinkage with an irregular outline, nuclear shrinkage with cloudy karyoplasm, and karyopyknosis in the late stage. Sections stained with Heidenhain’s iron hematoxylin method was used to pinpoint mitochondria and revealed cells which were undergoing the first stages of the apoptosis process i.e., disappearance of mitochondria from the cell, chromatin condensation and margination, paracentral localization of nucleoli, and vacuolated nuclei. In more advanced stages of apoptosis, cells presented significant nuclear and cytoplasmic changes. It was concluded that this is the first report targeting the mitochondria, by performing inexpensive histological staining techniques, in order to assess dead cells in situ.     

Identification of MEN1 gene mutations in sporadic carcinoid tumors of the lung

Lung carcinoids occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). There are no well defined genetic abnormalities known to occur in these tumors. We studied 11 sporadic lung carcinoids for loss of heterozygosity (LOH) at the locus of the MEN1 gene on chromosome 11q13, and for mutations of the MEN1 gene using dideoxy fingerprinting. Additionally, a lung carcinoid from a MEN1 patient was studied. In four of 11 (36%) sporadic tumors, both copies of the MEN1 gene were inactivated. All four tumors showed the presence of a MEN1 gene mutation and loss of the other allele. Observed mutations included a 1 bp insertion, a 1 bp deletion, a 13 bp deletion and a single nucleotide substitution affecting a donor splice site. Each mutation predicts truncation or potentially complete loss of menin. The remaining seven tumors showed neither the presence of a MEN1 gene mutation nor 11q13 LOH. The tumor from the MEN1 patient showed LOH at chromosome 11q13 and a complex germline MEN1 gene mutation. The data implicate the MEN1 gene in the pathogenesis of sporadic lung carcinoids, representing the first defined genetic alteration in these tumors.