Biomedical subjects

Frank Kirchhoff

Publications and source records attributed to Frank Kirchhoff.

At least 37 records · Page 2Linked to original sources

PubMed2005

Effect of naturally-occurring gp41 HR1 variations on susceptibility of HIV-1 to fusion inhibitors.

BACKGROUND: Sequence variations in the gp41 heptad repeat 1 (HR1) region have been identified in some treatment-naive HIV-1-infected patients but it remained elusive whether they confer resistance to fusion inhibitors. OBJECTIVE: To evaluate whether naturally occurring sequence variations in the HIV-1 group M gp41 HR1 region affect the sensitivity to inhibition by T-20 and T-1249. METHODS: Site-directed mutagenesis was used to generate HIV-1 NL4-3 mutants containing changes in the gp41 HR1 domain which have been previously identified in treatment-naive patients infected with various HIV-1 group M subtypes. HIV-1 variants were produced by transient transfection of 293T cells and used to determine viral infectivity and sensitivity to the fusion inhibitors T-20 and T-1249. RESULTS: Most naturally occurring sequence variations in the HR1 domain did not reduce viral infectivity. Three of the 10 HIV-1 variants analysed containing a single substitution of L33V, which is frequently present in subtype D isolates, or combined changes of L54M/Q56K or L34M/L54M/Q56R showed about fivefold reduced sensitivity to inhibition by T-20. In comparison, none of these HR1 sequence variations conferred resistance to T-1249. CONCLUSION: Some naturally occurring sequence variations in the gp41 HR1 region reduce sensitivity of HIV-1 to inhibition by T-20 but not T-1249.

Amino Acid Sequence↗

PubMed2005

The role of upstream U3 sequences in HIV-1 replication and CD4+ T cell depletion in human lymphoid tissue ex vivo.

The LTRs of all primate lentiviruses contain long U3 regions overlapping the nef gene. To assess the relevance of the modulatory U3 region for HIV-1 replication, we inactivated the T-rich region, the Polypurine tract and attachment (att) sequences in nef by silent mutations and inserted intact cis-regulatory elements just upstream of the core enhancer. These modifications severely truncated the U3 region and eliminated the nef overlap. The resulting HIV-1 mutants expressed functional Nef, replicated efficiently and caused CD4+ T cell depletion in ex vivo-infected lymphoid tissue suggesting that the modulatory U3 region might not be essential for efficient HIV-1 gene expression and AIDS pathogenesis.

CD4 Lymphocyte Count↗

PubMed2005

Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo.

Microglial cells represent the immune system of the mammalian brain and therefore are critically involved in various injuries and diseases. Little is known about their role in the healthy brain and their immediate reaction to brain damage. By using in vivo two-photon imaging in neocortex, we found that microglial cells are highly active in their presumed resting state, continually surveying their microenvironment with extremely motile processes and protrusions. Furthermore, blood-brain barrier disruption provoked immediate and focal activation of microglia, switching their behavior from patroling to shielding of the injured site. Microglia thus are busy and vigilant housekeepers in the adult brain.

Animals↗

PubMed2005

Expression of multidrug resistance proteins (Mrps) in astrocytes of the mouse brain: a single cell RT-PCR study.

Multidrug resistance proteins (Mrps) are ATP-driven export pumps which mediate the export of organic anions such as glutathione conjugates and glucuronides from eukaryotic cells. Within the central nervous system astrocytes have important functions in metabolism and detoxification. In such processes Mrps play essential roles. To identify the Mrp repertoire of mouse brain and of astrocytes in particular, the expression of six mouse Mrps was investigated by reverse-transcription polymerase-chain reaction (RT-PCR). Using mouse brain mRNA as source, amplification products were obtained for Mrp1, Mrp3, Mrp4, Mrp5 and Mrp6. In contrast, mRNA of Mrp2 could not be detected in mouse brain. To investigate whether individual astrocytes express different Mrps in brain, single-cell RT-PCRs were performed from the cytosol harvested from single astrocytes in acutely isolated brain slices from cortex and cerebellum of TgN(GFAP-EGFP) mice. In these mice astrocytes can readily be identified by glial fibrillary acidic protein promoter-controlled green fluorescent protein expression. Investigation of individual cortical astrocytes and Bergmann glial cells revealed that these cells express Mrp1, Mrp4 and Mrp5 and that individual astrocytes can contain mRNA of one, two or three of these Mrps simultaneously.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2005

Expression of reef coral fluorescent proteins in the central nervous system of transgenic mice.

Reef coral fluorescent proteins (RCFPs) are bright fluorescent proteins (FPs) covering a wide spectral range. We used various RCFP genes to transgenically color different cell populations in the brain. The mouse Thy1.2 promoter was used to target expression of HcRed1 in neurons, the human glial fibrillary acidic protein (GFAP) promoter to label astrocytes with AmCyan1, AsRed2 and mRFP1 as well as the mouse proteolipid protein promoter to mark oligodendrocytes with DsRed1. In brain sections of transgenic mice, RCFP expression was found to be highly specific using immunohistochemistry and fluorescence microscopy. In contrast to transgenic mice with expression of jellyfish FP variants, RCFPs formed numerous fluorescent precipitates. These aggregates were primarily found in cell somata and also in cell processes. Older mice were more affected than younger ones. Despite these fluorescent deposits, physiological properties of RCFP expressing brain cells such as whole-cell membrane currents or glutamate-evoked calcium signaling seemed to be unaffected. While brightness and spectral variation of RCFPs are optimal for expression in transgenic animals used in physiological experiments, the formation of fluorescent precipitates in various cell types limits their use for morphological cell analysis in situ.

Age Factors↗

PubMed2005

Nef induces multiple genes involved in cholesterol synthesis and uptake in human immunodeficiency virus type 1-infected T cells.

Several recent reports indicate that cholesterol might play an important role in human immunodeficiency virus type 1 (HIV-1) replication. We investigated the effects of HIV-1 infection on cholesterol biosynthesis and uptake using microarrays. HIV-1 increased gene expression of cholesterol genes in both transformed T-cell lines and primary CD4(+) T cells. Consistent with our microarray data, (14)C-labeled mevalonate and acetate incorporation was increased in HIV-1-infected cells. Our data also demonstrate that changes in cholesterol biosynthesis and uptake are only observed in the presence of functional Nef, suggesting that increased cholesterol synthesis may contribute to Nef-mediated enhancement of virion infectivity and viral replication.

Aspartic Acid Endopeptidases↗

PubMed2005

Primary sooty mangabey simian immunodeficiency virus and human immunodeficiency virus type 2 nef alleles modulate cell surface expression of various human receptors and enhance viral infectivity and replication.

The nef gene of the pathogenic simian immunodeficiency virus (SIV) mac239 clone has been well characterized. Little is known, however, about the function of nef alleles derived from naturally SIVsm-infected sooty mangabeys (Cercocebus atys) and from human immunodeficiency virus type 2 (HIV-2)-infected individuals. Addressing this, we demonstrate that, similarly to the SIVmac239 nef, primary SIVsm and HIV-2 nef alleles down-modulate cell surface expression of human CD4, CD28, CD3, and class I or II major histocompatibility complex (MHC-I or MHC-II, respectively) molecules, up-regulate surface expression of the invariant chain (Ii) associated with immature MHC-II, inhibit early T-cell activation events, and enhance virion infectivity. Both also stimulate viral replication, although HIV-2 nef alleles were less active in this assay than SIVsm nef alleles. Mutational analysis showed that a dileucine-based sorting motif in the C-proximal loop of SIV or HIV-2 Nef is critical for its effects on CD4, CD28, and Ii but dispensable for down-regulation of CD3, MHC-I, and MHC-II. The C terminus of SIV and HIV-2 Nef was exclusively required for down-modulation of MHC-I, further demonstrating that analogous functions are mediated by different domains in Nef proteins derived from different groups of primate lentiviruses. Our results demonstrate that none of the eight Nef functions investigated had been newly acquired after cross-species transmission of SIVsm from naturally infected mangabeys to humans or macaques. Notably, HIV-2 and SIVsm nef alleles efficiently down-modulate CD3 and C28 surface expression and inhibit T-cell activation more efficiently than HIV-1 nef alleles. These differences in Nef function might contribute to the relatively low levels of immune activation observed in HIV-2-infected human individuals.

Alleles↗

PubMed2005

Nef proteins from diverse groups of primate lentiviruses downmodulate CXCR4 to inhibit migration to the chemokine stromal derived factor 1.

Nef proteins of primate lentiviruses promote viral replication, virion infectivity, and evasion of antiviral immune responses by modulating signal transduction pathways and downregulating expression of receptors at the cell surface that are important for efficient antigen-specific responses, such as CD4, CD28, T-cell antigen receptor, and class I and class II major histocompatibility complex. Here we show that Nef proteins from diverse groups of primate lentiviruses which do not require the chemokine receptor CXCR4 for entry into target cells strongly downmodulate the cell surface expression of CXCR4. In contrast, all human immunodeficiency virus type 1 (HIV-1) and the majority of HIV-2 Nef proteins tested did not have such strong effects. SIVmac239 Nef strongly inhibited lymphocyte migration to CXCR4 ligand, the chemokine stromal derived factor 1 (SDF-1). SIVmac239 Nef downregulated CXCR4 by accelerating the rate of its endocytosis. Downmodulation of CXCR4 was abolished by mutations that disrupt the constitutively strong AP-2 clathrin adaptor binding element located in the N-terminal region of the Nef molecule, suggesting that Nef accelerates CXCR4 endocytosis via an AP-2-dependent pathway. Together, these results point to CXCR4 as playing an important role in simian immunodeficiency virus and possibly also HIV-2 persistence in vivo that is unrelated to viral entry into target cells. We speculate that Nef targets CXCR4 to disrupt ordered trafficking of infected leukocytes between local microenvironments in order to facilitate their dissemination and/or impair the antiviral immune response.

CD4 Antigens↗

PubMed2005

Human immunodeficiency virus type 1 inhibits DNA damage-triggered apoptosis by a Nef-independent mechanism.

It is controversial whether the accessory human immunodeficiency virus type 1 (HIV-1) Nef protein inhibits or enhances apoptosis. To address this issue, we investigated the effect of Nef on programmed cell death with vectors or proviral HIV-1 constructs coexpressing Nef and green fluorescent protein from single bicistronic RNAs. This approach allows us to readily identify transfected or infected cells and to correlate cell death directly with Nef expression levels. We demonstrate that Nef does not significantly affect apoptosis in transfected or HIV-1-infected Jurkat T cells or primary human peripheral blood mononuclear cells. Unexpectedly, however, both nef+ and nef-defective HIV-1 infection blocked apoptosis in cells treated with UV light or etoposide but not cell death induced by CD95 antibody, TRAIL, Ly294002, or serum starvation. Our results show that HIV-1 infection inhibits DNA damage-induced but not death receptor-dependent cell death by a Nef-independent mechanism.

Apoptosis↗

PubMed2004

Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo.

Glial cells have been identified as key signaling components in the brain; however, methods to investigate their structure and function in vivo have been lacking. Here, we describe a new, highly selective approach for labeling astrocytes in intact rodent neocortex that allows in vivo imaging using two-photon microscopy. The red fluorescent dye sulforhodamine 101 (SR101) was specifically taken up by protoplasmic astrocytes after brief exposure to the brain surface. Specificity was confirmed by immunohistochemistry. In addition, SR101 labeled enhanced green fluorescent protein (EGFP)-expressing astrocytes but not microglial cells in transgenic mice. We used SR101 labeling to quantify morphological characteristics of astrocytes and to visualize their close association with the cortical microvasculature. Furthermore, by combining this method with calcium indicator loading of cell populations, we demonstrated distinct calcium dynamics in astroglial and neuronal networks. We expect SR101 staining to become a principal tool for investigating astroglia in vivo.

Animals↗

PubMed2004

Differential regulation of human immunodeficiency virus type 2 and simian immunodeficiency virus promoter activity.

Promoter activity of the HIV-1 long terminal repeat (LTR) is largely dependent on intact NF-kB and SpI binding sites in the U3 region. In contrast, upstream LTR sequences allow efficient simian immunodeficiency virus (SIVmac) transcription in the absence of the core enhancer promoter region. In the present study, we investigated whether the regulation of HIV-2 Rod LTR activity is more reminiscent of HIV-1 having the same host or of SIVmac239 belonging to the same phylogenetic group. Viral promoter activity was studied in the context of the integrated provirus using both single cycle assays with pseudotyped luciferase reporter viruses and replication-competent HIV-2 LTR mutants. Our results demonstrate that intact SpI binding sites are important for both HIV-2 and SIVmac LTR activity in T cells and monocyte-derived macrophages. In contrast, deletion of the NF-kB binding site or of upstream regulatory sequences impaired HIV-2 Rod LTR activity but had little effect on SIVmac239 promoter function. Thus, similar to HIV-1, regulation of HIV-2 LTR promoter activity shows a low degree of functional redundancy possibly suggesting a specific adaptation to the human host.

Binding Sites↗

PubMed2004

Diversity of functional astroglial properties in the respiratory network.

A population of neurons in the caudal medulla generates the rhythmic activity underlying breathing movements. Although this neuronal network has attracted great attention for studying neuronal aspects of synaptic transmission, functions of glial cells supporting this neuronal activity remain unclear. To investigate the role of astrocytes in the respiratory network, we applied electrophysiological and immunohistochemical techniques to characterize astrocytes in regions involved in the generation and transmission of rhythmic activity. In the ventral respiratory group and the hypoglossal nucleus (XII) of acutely isolated brainstem slices, we analyzed fluorescently labeled astrocytes obtained from TgN(GFAP-EGFP) transgenic mice with the whole-cell voltage-clamp technique. Three subpopulations of astrocytes could be discerned by their distinct membrane current profiles. A first group of astrocytes was characterized by nonrectifying, symmetrical and voltage-independent potassium currents and a robust glutamate transporter response to d-aspartate. A second group of astrocytes showed additional A-type potassium currents, whereas a third group, identified by immunolabeling for the glial progenitor marker NG2, expressed outwardly rectifying potassium currents, smaller potassium inward currents, and only minimal D-aspartate-induced transporter currents. Astrocytes of all groups showed kainate-induced inward currents. We conclude that most of the astrocytes serve as a buffer system of excess extracellular glutamate and potassium; however, a distinct cell population (NG2-positive, A-type potassium currents) may play an important role for network plasticity.

Amino Acid Transport System X-AG↗

PubMed2004

Inhibitory effect of the lectin wheat germ agglutinin on the binding of 125I-CCK-8s to the CCK-A and -B receptors of AR42J cells.

INTRODUCTION: Cholecystokinin (CCK) is a peptide hormone and plays a major role both in the regulation of pancreatic enzyme secretion and growth of the gastrointestinal tract. The pancreatic CCK receptors are highly glycosylated membrane proteins that are able to bind plant lectins such as wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA-I). AIM AND METHODOLOGY: In preceding papers, we demonstrated an inhibition of CCK-8s induced Ca2+ signaling and secretion of rat pancreatic acini and AR42J cells by the lectins WGA and UEA-I (Pancreas 2001;23:368-374). Here we studied the influence of WGA, UEA-I, and 22 other lectins on 125I-CCK-8s binding on AR42J cells. A binding assay was used with 125I-CCK-8s and dexamethasone-stimulated AR42J cells, bearing CCK-A as well as CCK-B receptors. RESULTS: WGA inhibits 125I-CCK-8s binding in a dose-dependent manner. The binding is affected at concentrations of WGA >1 microg/mL. The EC50 for inhibition is 8 microg/mL. At a concentration of 25 microg/mL, WGA inhibits the hormone binding 70%. This inhibition can be abolished by the specific sugars for WGA N,N',N"-triacetylchitotriose and N-acetylglucosamine, but not by N-acetylneuraminic acid. UEA-I diminished hormone binding but without significance, although UEA-I binds to the fucose residues of receptor glycosylations. All other 22 lectins tested here were ineffective. CONCLUSION: The blockage of CCK receptors by WGA explains the inhibition of CCK-8s induced Ca2+ signaling and the secretion of pancreatic acinar cells and AR42J cells. Although the inhibitory effect of WGA is in agreement with the findings of Santer et al, the results with UEA-I are in contrast to those of Santer et al (1990), who described a strong increase in 125I-CCK-8s binding to isolated crude rat pancreatic cell membranes in the presence of UEA-I.

Acetylglucosamine↗

PubMed2004

Astroglial processes show spontaneous motility at active synaptic terminals in situ.

Within the tripartite structure of vertebrate synapses, enwrapping astroglial processes regulate synaptic transmission by transmitter uptake and by direct transmitter release. We applied confocal and two-photon laser scanning microscopy to acutely isolated slices prepared from the brainstem of transgenic TgN(GFAP-EGFP) mice. In transversal sections fluorescently labelled astrocytes are evenly distributed throughout the tissue. Astroglial processes contacted neuronal somata and enwrapped active synaptic terminals as visualized using FM1-43 staining in situ. Here, at these synaptic regions astroglial process endings displayed a high degree of dynamic morphological changes. Two defined modes of spontaneous motility could be distinguished: (i) gliding of thin lamellipodia-like membrane protrusions along neuronal surfaces and (ii) transient extensions of filopodia-like processes into the neuronal environment. Our observations highlight the active role of astrocytes in direct modulation of synaptic transmission.

Animals↗

PubMed2004

Nef proteins from simian immunodeficiency virus-infected chimpanzees interact with p21-activated kinase 2 and modulate cell surface expression of various human receptors.

The accessory Nef protein allows human immunodeficiency virus type 1 (HIV-1) to persist at high levels and to cause AIDS in infected humans. The function of HIV-1 group M subtype B nef alleles has been extensively studied, and a variety of in vitro activities believed to be important for viral pathogenesis have been established. However, the function of nef alleles derived from naturally simian immunodeficiency virus (SIV)-infected chimpanzees, the original host of HIV-1, or from the HIV-1 N and O groups resulting from independent zoonotic transmissions remains to be investigated. In the present study we demonstrate that SIVcpz and HIV-1 group N or O nef alleles down-modulate CD4, CD28, and class I or II MHC molecules and up-regulate surface expression of the invariant chain (Ii) associated with immature major histocompatibility complex (MHC) class II. Furthermore, the ability of Nef to interact with the p21-activated kinase 2 was generally conserved. The functional activity of HIV-1 group N and O nef genes did not differ significantly from group M nef alleles. However, SIVcpz nef genes as a group showed a 1.8- and 2.0-fold-higher activity in modulating CD28 (P = 0.0002) and Ii (P = 0.016) surface expression, respectively, but were 1.7-fold less active in down-regulating MHC class II molecules (P = 0.006) compared to HIV-1 M nef genes. Our finding that primary SIVcpz nef alleles derived from naturally infected chimpanzees modulate the surface expression of various human cellular receptors involved in T-cell activation and antigen presentation suggests that functional nef genes helped the chimpanzee virus to persist efficiently in infected humans immediately after zoonotic transmission.

Adult↗

PubMed2004

A naturally occurring variation in the proline-rich region does not attenuate human immunodeficiency virus type 1 nef function.

We analyzed human immunodeficiency virus type 1 (HIV-1) Nef variants to further evaluate the functional relevance of the R71T substitution previously proposed to attenuate viral replication (Fackler et al., Curr. Biol. 11:1294-1299, 2001). Our results demonstrate that this variation in the proline-rich region does not significantly affect the functional activity of Nef or HIV-1 infectivity or replication.

Amino Acid Substitution↗

PubMed2004

Comprehensive analysis of nef functions selected in simian immunodeficiency virus-infected macaques.

A variety of simian immunodeficiency virus (SIVmac) nef mutants have been investigated to clarify which in vitro Nef functions contribute to efficient viral replication and pathogenicity in rhesus macaques. Most of these nef alleles, however, were only functionally characterized for their ability to down-modulate CD4 and class I major histocompatibility complex (MHC-I) cell surface expression and to enhance SIV replication and infectivity. To obtain information on the in vivo relevance of more recently established Nef functions, we examined the ability of a large panel of constructed SIVmac Nef mutants and of variants that emerged in infected macaques to down-regulate CD3, CD28, and MHC-II and to up-regulate the MHC-II-associated invariant chain (Ii). We found that all these four Nef functions were restored in SIV-infected macaques. In most cases, however, the initial mutations and the changes selected in vivo affected several in vitro Nef functions. For example, truncated Nef proteins that emerged in animals infected with SIVmac239 containing a 152-bp deletion in nef efficiently modulated both CD3 and Ii surface expression. Overall, our results suggest that the effect of Nef on each of the six cellular receptors investigated contributes to viral fitness in the infected host but also indicate that modulation of CD3, MHC-I, MHC-II, or Ii surface expression alone is insufficient for SIV virulence.

Animals↗

PubMed2004

Vpr and Vpu are important for efficient human immunodeficiency virus type 1 replication and CD4+ T-cell depletion in human lymphoid tissue ex vivo.

The relevance of the accessory vpr, vpu, and nef genes for human immunodeficiency virus type 1 (HIV-1) replication in human lymphoid tissue (HLT), the major site of viral replication in vivo, is largely unknown. Here, we show that an individual deletion of nef, vpr, or vpu significantly decreases HIV-1 replication and prevents CD4+ T-cell depletion in ex vivo HLT. However, only combined defects in all three accessory genes entirely disrupt the replicative capacity of HIV-1. Our results demonstrate that nef, vpr, and vpu are all essential for efficient viral spread in HLT, suggesting an important role in AIDS pathogenesis.

CD4-Positive T-Lymphocytes↗