Perturbation of Host Nuclear Membrane Component RanBP2 Impairs the Nuclear Import of Human Immunodeficiency Virus -1 Preintegration Complex (DNA)

HIV-1 is a RNA virus that requires an intermediate DNA phase via reverse transcription (RT) step in order to establish productive infection in the host cell. The nascent viral DNA synthesized via RT step and the preformed viral proteins are assembled into pre-integration complex (PIC) in the cell cytoplasm. To integrate the viral DNA into the host genome, the PIC must cross cell nuclear membrane through the nuclear pore complex (NPC). RanBP2, also known as Nup358, is a major component of the cytoplasmic filaments that emanates from the nuclear pore complex and has been implicated in various nucleo-cytoplasmic transport pathways including those for HIV Rev-protein. We sought to investigate the role of RanBP2 in HIV-1 replication. In our investigations, we found that RanBP2 depletion via RNAi resulted in profound inhibition of HIV-1 infection and played a pivotal role in the nuclear entry of HIV DNA. More precisely, there was a profound decline in 2-LTR DNA copies (marker for nuclear entry of HIV DNA) and an unchanged level of viral reverse transcription in RanBP2-ablated HIV-infected cells compared to RanBP3-depleted or non-specific siRNA controls. We further demonstrated that the function of Rev was unaffected in RanBP2-depleted latently HIV infected cells (reactivated). We also serendipitously found that RanBP2 depletion inhibited the global ectopic gene expression. In conclusion, RanBP2 is a host factor that is involved in the nuclear import of HIV-1 PIC (DNA), but is not critical to the nuclear export of the viral mRNAs or nucleo-cytoplasmic shuttling of Rev. RanBP2 could be a potential target for efficient inhibition of HIV

The macrophage in HIV-1 infection: From activation to deactivation?

Macrophages play a crucial role in innate and adaptative immunity in response to microorganisms and are an important cellular target during HIV-1 infection. Recently, the heterogeneity of the macrophage population has been highlighted. Classically activated or type 1 macrophages (M1) induced in particular by IFN-γ display a pro-inflammatory profile. The alternatively activated or type 2 macrophages (M2) induced by Th-2 cytokines, such as IL-4 and IL-13 express anti-inflammatory and tissue repair properties. Finally IL-10 has been described as the prototypic cytokine involved in the deactivation of macrophages (dM). Since the capacity of macrophages to support productive HIV-1 infection is known to be modulated by cytokines, this review shows how modulation of macrophage activation by cytokines impacts the capacity to support productive HIV-1 infection. Based on the activation status of macrophages we propose a model starting with M1 classically activated macrophages with accelerated formation of viral reservoirs in a context of Th1 and proinflammatory cytokines. Then IL-4/IL-13 alternatively activated M2 macrophages will enter into the game that will stop the expansion of the HIV-1 reservoir. Finally IL-10 deactivation of macrophages will lead to immune failure observed at the very late stages of the HIV-1 disease

Host hindrance to HIV-1 replication in monocytes and macrophages

Monocytes and macrophages are targets of HIV-1 infection and play critical roles in multiple aspects of viral pathogenesis. HIV-1 can replicate in blood monocytes, although only a minor proportion of circulating monocytes harbor viral DNA. Resident macrophages in tissues can be infected and function as viral reservoirs. However, their susceptibility to infection, and their capacity to actively replicate the virus, varies greatly depending on the tissue localization and cytokine environment. The susceptibility of monocytes to HIV-1 infection in vitro depends on their differentiation status. Monocytes are refractory to infection and become permissive upon differentiation into macrophages. In addition, the capacity of monocyte-derived macrophages to sustain viral replication varies between individuals. Host determinants regulate HIV-1 replication in monocytes and macrophages, limiting several steps of the viral life-cycle, from viral entry to virus release. Some host factors responsible for HIV-1 restriction are shared with T lymphocytes, but several anti-viral mechanisms are specific to either monocytes or macrophages. Whilst a number of these mechanisms have been identified in monocytes or in monocyte-derived macrophages in vitro, some of them have also been implicated in the regulation of HIV-1 infection in vivo, in particular in the brain and the lung where macrophages are the main cell type infected by HIV-1. This review focuses on cellular factors that have been reported to interfere with HIV-1 infection in monocytes and macrophages, and examines the evidences supporting their role in vivo, highlighting unique aspects of HIV-1 restriction in these two cell types

Bepaling van chloramphenicol in runder-urine, vlees en garnalen m.b.v. GC-MS. Methode validatie volgens Commissie Beschikking 2002/657/EC

This report describes the validation of the quantification and the identification of an analytical method for the determination of low concentrations (0.1-1.0 micro g/kg) of chloramphenicol in samples of urine, shrimps and meat. The validation study was based on the criteria described in Decision 2002/657/EC of the European Commission. The analytical method consists of an enzymatic hydrolysis (urine) or enzymatic digestion (meat), followed by liquid-liquid extraction of chloramphenicol from the matrix with ethyl acetate. The extract is cleaned with Solid Phase Extraction (SPE), followed by LC fractionation. The SPE step can be omitted for shrimps. After derivatisation of the chloramphenicol, final separation and detection is performed with GC-MS with Negative Chemical Ionisation (NCI). Detection can also be carried out using Electron Impact (EI), which is a less sensitive technique. This method can be used for both screening and quantification. The limit of determination for all samples is approximately 0.05 micro g/l or micro g/kg. The detection capability for samples of urine is 0.3 micro g/l. For shrimp samples, the detection capability is 0.1 micro g/kg. If EI is used, the detection capability is 0.5micro g/l or micro g/kg.Dit rapport beschrijft de validatie, de kwantificering en de wijze van identificatie van een analysemethode voor de bepaling van lage concentraties (0,1-1,0 micro g/kg) chlooramphenicol in monsters urine, garnalen en spierweefsel (vlees) Deze validatie is gebaseerd op de criteria beschreven in de Beschikking van de Commissie 2002657EC. Na een eerste extractie, voorafgegaan door enzymatische hydrolyse (urine) of enzymatische digestie (spierweefsel), wordt chlooramphenicol geextraheerd vanuit de matrix met ethylacetaat. Het verkregen extract wordt vervolgens verder gezuiverd met vaste fase extractie (Solid Phase Extraction, SPE) en LC-fractionering. Bij het opwerken van monsters garnaal kan de zuiveringsstap over SPE worden overgeslagen. Na derivatisering wordt het verkregen extract geanalyseerd met GC-MS. Detectie kan plaatsvinden met negatieve chemische ionisatie (NCI), de meest gevoelige methode. Indien NCI niet beschikbaar is kunnen electron impact (EI) of positieve chemische ionisatie (PCI) als alternatief gebruikt worden. De beschreven methode is zowel geschikt voor screening als bevestiging. De beslissingsgrens voor alle monsters bedraagt ongeveer 0,05 micro g/l of 0,05 micro g/kg. Het detectievermogen voor urinemonsters is 0,3 micro g/l, voor garnalen is deze 0,1 micro g/kg. Wanneer PCI of EI gebruikt worden is het detectievermogen 0,5 microg/l of 0,5 micro g/kg

Bepaling van chloramphenicol in runder-urine, vlees en garnalen m.b.v. GC-MS. Methode validatie volgens Commissie Beschikking 2002/657/EC

Dit rapport beschrijft de validatie, de kwantificering en de wijze van identificatie van een analysemethode voor de bepaling van lage concentraties (0,1-1,0 micro g/kg) chlooramphenicol in monsters urine, garnalen en spierweefsel (vlees) Deze validatie is gebaseerd op de criteria beschreven in de Beschikking van de Commissie 2002657EC. Na een eerste extractie, voorafgegaan door enzymatische hydrolyse (urine) of enzymatische digestie (spierweefsel), wordt chlooramphenicol geextraheerd vanuit de matrix met ethylacetaat. Het verkregen extract wordt vervolgens verder gezuiverd met vaste fase extractie (Solid Phase Extraction, SPE) en LC-fractionering. Bij het opwerken van monsters garnaal kan de zuiveringsstap over SPE worden overgeslagen. Na derivatisering wordt het verkregen extract geanalyseerd met GC-MS. Detectie kan plaatsvinden met negatieve chemische ionisatie (NCI), de meest gevoelige methode. Indien NCI niet beschikbaar is kunnen electron impact (EI) of positieve chemische ionisatie (PCI) als alternatief gebruikt worden. De beschreven methode is zowel geschikt voor screening als bevestiging. De beslissingsgrens voor alle monsters bedraagt ongeveer 0,05 micro g/l of 0,05 micro g/kg. Het detectievermogen voor urinemonsters is 0,3 micro g/l, voor garnalen is deze 0,1 micro g/kg. Wanneer PCI of EI gebruikt worden is het detectievermogen 0,5 microg/l of 0,5 micro g/kg.This report describes the validation of the quantification and the identification of an analytical method for the determination of low concentrations (0.1-1.0 micro g/kg) of chloramphenicol in samples of urine, shrimps and meat. The validation study was based on the criteria described in Decision 2002/657/EC of the European Commission. The analytical method consists of an enzymatic hydrolysis (urine) or enzymatic digestion (meat), followed by liquid-liquid extraction of chloramphenicol from the matrix with ethyl acetate. The extract is cleaned with Solid Phase Extraction (SPE), followed by LC fractionation. The SPE step can be omitted for shrimps. After derivatisation of the chloramphenicol, final separation and detection is performed with GC-MS with Negative Chemical Ionisation (NCI). Detection can also be carried out using Electron Impact (EI), which is a less sensitive technique. This method can be used for both screening and quantification. The limit of determination for all samples is approximately 0.05 micro g/l or micro g/kg. The detection capability for samples of urine is 0.3 micro g/l. For shrimp samples, the detection capability is 0.1 micro g/kg. If EI is used, the detection capability is 0.5micro g/l or micro g/kg.VWA Kv

Monitoring of dioxines in cow's milk in riskareas

In this paper, results are reported of a monitoring sutdy on levels of PCDDs and PCDFs in cow's milk from dairy farms in the vicinity of three municipal solid waste incinerators (MSWs) and one metal reclamation plant in the Netherlands. Levels are expressed in picograms 2,3,7,8- TCDD toxicity equivalents (TEQ) per gram of milk fat, calculated on the basis of international toxicity equivalence factors (TEF) of individual components. In June sampling near the MSW of Alkmaar recommenced after the improvement of the incinerator. Samples consisted of time-averaged sampling of cow's milk during periods of one month of in total thirteen dairy farms near Alkmaar, Duiven, in the Lickebaert-area and in Culemborg. Samples were collected in the period from May to June 1991.HIMHHIGBVV

Monitoring of dixoins in cow's milk in riskareas. Section IV

In this paper, results are reported of a monitoring study on levels of PCDDs and PCDFs in cow's milk from dairy farms in the vicinity of two municipal solid waste incinerators (MSWs) and a metal recelamation plant in the Netherlands. Levels are expressed in picograms 2,3,7,8- TCDD toxicity equivalents (TEQ) per gram of milk fat, calculated on the basis of international toxicity equivalence factors (I-TEF) of individual components. Samples consisted of time-averaged sampling of cow's milk during periods of one month of in total eight dairy farms in Duiven, in the Lickebaert-area and in Culemborg. Samples were collected in the period from January to April 1991. The following conclusions have been drawn. 1. In the area near the MSW at Duiven, dioxin levels were between 2.2+-0.1 and 5.9+-0.3 and remained below the Dutch legislation level for dioxins in cow's milk (6 pg TEQ/g fat). Mean dioxin level in cow's milk of the two dairy farms in Duiven during this period (3.3pg TEQ/g of milk fat) were lower than the mean of the previous period (3.9pg TEQ/g of fat). 2.2 In the vicinity of the metal reclamation plant in Culemborg, levels ranged between 5.9 +- 03 and 2.8 +- 0.1 pg TEQ/g of fat. The dioxin level in this area also remains below the Dutch legislation level for dioxins in cow's milk. The mean dioxin level (4.4 pg TEQ/g of milk fat) in cow's milk of this dairy farm was comparable with the mean level of the previous period (4.3 pg TEQ/g of fat). 3. Levels of dioxins in cow's milk in the Lickebaert area were between 4.4 +- 0.2 and 11.0 +- 0.6 pg TEQ/g of fat. The mean level of five dairy farms (5.9 pg TEQ/g of milk fat) during the period January-April 1991 was lower than the corresponding mean in the previous period (7.9 pg TEQ/g of fat). It is not clear whether this decline is incidental or structural.HIMHHIGBVV

Monitoring of dioxins in cow's milk in Zeeuws-Vlaanderen

Because of the presence of municipal waste incinerators (MSWs) just across the Dutch-Belgian border in Zeeuws-Vlaanderen, the Netherlands, elevated dioxin levels in cow's milk were expected in this region. From September 3 to December 4, 1991, milk samples were collected from six dairy farms near Sluis, Zeeuws-Vlaanderen, and analysed for PCDDs and PCDFs From each farm, milk samples collected during periods on one month were pooled to monthly averaged samples prior to analysis. Dioxin levels in montly averaged milk samples from six diary farms in Zeeuws-Vlaanderen in the neighbourhood of the Belgian MSW ranged from 1.2+/-0.1 to 5.9+/-0.3 pg TEQ/g of fat. These levels are elevated when compared to the background level for dioxins in cow's milk (0.8-2.5 pg TEQ/g of fat) in the Netherlands. In milk from dairy farm Go-C, with its pastures closest to the incinerator at Westkapelle, the highest levels were found (3.5+/-0.2 and 5.9+/-0.3 pg TEQ/g of fat, respectively). It cannot be excluded that levels in the milk from this dairy farm, may occasionally exceed the level of 6 pg TEQ/g of fat, the current legislation level for dioxins in cow's milk. The congener pattern in cow's milk from the dairy farm Go-C differed from the usual pattern observed in cow's milk sampled in the vicinity of MSWs. It is not yet clear whether this difference is due to the emissions of the Belgian MSW or other possible sources of dioxins.HIHMHIGBVV

Monitoring van dioxinen in koemelk in risicogebieden. Deelrapport III

In this paper, results are reported of a monitoring study on levels of PCDDs and PCDFs in cow's milk from dairy farms in the vicinity of municipal solid waste incinerators (MSWs) and a metal reclamation plant in the Netherlands. Levels are expressed in picograms of 2,3,7,8- TCDD toxicity equivalents (TEQ) per gram of milk fat, calculated on the basis of international toxicity equivalence factors (TEF) of individual components. Samples consisted of time-averaged sampling of cow's milk during periods of one month of in total 12 dairy farms in the neighbourhood of the MSWs of Duiven, Zaandam and the Lickebaert- area and a farm near the metal reclamation plant of Culemborg. Samples were collected in the period from September to December 1990.<br

[Monitoring of dioxins in cow&apos;s milk in areas of risk. Part XI.]

Abstract niet beschikbaarThis report presents the results of the monitoring of dioxins in cow's milk from selected dairy farms. These dairy farms are located near the municipal waste incinerator (MWI) of Rotterdam (the Lickebaert area), the Netherlands. Levels are expressed in picograms 2,3,7,8-TCDD toxicity equivalents (TEQ) per gram of milk fat, calculated on the basis of international toxicity equivalence factors (I-TEF) of individual congeners. Now, the levels in cow's milk of the months January to March are known. In this report the dioxin levels in the Lickebaert area are presented for the months February 1990 to March 1993. The increase of the dioxin levels observed in the preceding period from July on, has apparently reached a maximum in September 1992 at a level of 8.9-13.3 pg TEQ/g of fat. The levels of the following months show a slight decrease. In the months October to December 1992 the dioxin levels in the milk samples ranged between 6.7 and 10.9 pg TEQ/g of fat. In the months January to March 1993 the levels ranged between 6.4 and 10.0 pg TEQ/g of fat and are comparable to the levels of the months October to December 1992. These levels still exceed the Dutch legislationlevel for dioxins in cow's milk.HIG