Summary
Peripheral blood mononuclear cells (PBMC) of untreated, HIV‐infected patients contain HIV‐specific CD8 T cells as well as their corresponding targets, HIV‐infected CD4 T cells. To determine ...if CD4 T‐cell depletion in HIV‐infected patients may result from autologous CD8–CD4 T‐cell interaction, CD8 and CD4 T cells procured from PBMC of acute and chronic untreated HIV‐infected patients were sorted and co‐incubated. Formation of CD8‐CD4 T‐cell conjugates was observed by fluorescence microscopy. Apoptosis of CD4 T cells in conjugation was recorded by digitized images and was further observed and measured by FACS using Annexin staining. Perforin expression in the CD8 T cells was measured using intracellular monoclonal perforin antibody staining. HIV DNA in the conjugated CD4 T cells was detected by in situ PCR. We found that 6·1 ± 0·5% of CD4 T cells from acute HIV‐infected patients and 3·0 ± 0·5% from chronic HIV‐infected patients formed CD8–CD4 T‐cell conjugates. Annexin binding and cell morphology typical of apoptosis were observed in the conjugated CD4 T cells. The majority of CD8 T cells that had conjugated to CD4 T cells expressed perforin. The conjugated CD4 T cells exhibited nuclear HIV DNA. CD8 T cells and HIV‐infected CD4 T cells, both procured from the PBMC of untreated HIV‐infected patients, form conjugates. Apoptotic lytic activity has been observed in the conjugated CD4 T cells. We propose that CD4 T‐cell annihilation in HIV‐infected patients results, at least in part, from the interactions of perforin‐rich CD8 T cells with autologous, HIV‐infected CD4 T cells.
•Israeli tap water's iodine decreased vs. prior data.•Low tap water iodine linked to low Mg, high Ca/Mg ratio, and low hardness.•Tap water supplies only 3.39% of RDA level.•Post-treated desalinated ...water meets industrial specs but lacks iodine and Mg.•Lack of drinking water iodine regs needs policy investigation.
In Israel, desalinated water is a major source of drinking water. Previous studies have suggested that the levels of iodine in water provided by authorities may not accurately reflect the levels reaching end-users.
We analyzed 21 tap water samples collected from different localities across Israel, 13 post-treated desalinated water samples from three of the largest Israeli desalination plants, and several natural water samples. An improved method of ICP-MS developed in our laboratory was used to analyze the content of iodine and other macro-elements, and determination of iodine was performed in alkaline media.
Our results showed that it is possible to distinguish between sample groups based on iodine concentration, water hardness, and Ca/Mg ratio. The median iodine concentrations for four groups of tap water samples ranged from 0.3 to 12.3 µg/L, which is lower than the concentrations previously reported by other researchers in Israel. Based on typical consumption, the water samples can provide no more than 3.39% of the recommended dietary allowance level for iodine. The analysis of post-treated desalinated water samples indicated that these waters comply with industrial specifications but contain only trace concentrations of iodine and much less magnesium than recommended by different public health authorities for public consumption of drinking water.
The total iodine concentrations found were lower than several observations reported in previous years in the literature. There are currently no strict regulations regarding iodine and magnesium levels in drinking and/or softened (desalinated) water, but the intensive desalination plant application is already exhibiting a negative impact on public health. Further investigations are needed, but the present study provides useful insights for developing an effective policy to ensure adequate iodine supply for the population of Israel through drinking water.
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The role of HIV-specific CD8 T cell activity in the course of HIV infection and the way it affects the virus that resides in the latent reservoir resting memory cells is debated. The PBMC of ...HIV-infected patients contain HIV-specific CD8 T cells and their potential targets, CD4 T cells latently infected by HIV. CD4 T cells and CD8 T cells procured from PBMC of HIV-infected patients were co-incubated and analyzed: Formation of CD8 T cells and HIV-infected CD4 T cell conjugates and apoptosis of these CD4 T cells were observed by fluorescence microscopy with
PCR of HIV LTR DNA. Furthermore, conjugation of CD8 T cells with CD4 T cells and apoptosis of CD4 T cells was observed and quantified by imaging flow cytometry using anti-human activated caspase 3 antibody and TUNEL assay. The conjugation activity and apoptosis were found to be much higher in patients with acute HIV infection or AIDS compared to patients in chronic infection on antiretroviral therapy (ART) or not. Patients on ART had low grade conjugation and apoptosis of isolated CD69, CD25, and HLA-DR-negative CD4 T cells (latent reservoir cells) by CD8 T cells. Using
PCR The latent reservoir CD4 T cells were shown to contain most of the HIV DNA. We demonstrate in HIV-infected patients, that CD8 T cells conjugate with and kill HIV-infected CD4 T cells, including HIV-infected resting memory CD4 T cells, throughout the course of HIV infection. We propose that in HIV-infected patients CD4 T cell annihilation is caused in part by ongoing activity of HIV-specific CD8 T cells. HIV Nef protein interacts with ASK 1 and inhibits its pro-apoptotic death signaling by Fas/FasL, thus protecting HIV-infected cells from CD8 T cells killing. A peptide that interrupts Nef-ASK1 interaction that had been delivered into CD4 T cells procured from patients on ART resulted in the increase of their apoptosis inflicted by autologous CD8 T cells. We suggest that elimination of the HIV-infected latent reservoir CD4 T cells can be achieved by Nef inhibition.
Reliable iodine determination in drinking water samples has gained importance in the last few decades, mostly due to intensive use of both desalinized water that lacks several important nutritional ...elements, and bottled mineral water. ICP-MS is a sensitive method for iodine determination that must be performed under alkaline conditions because of the volatile nature of some iodine species. However, in water samples with high pH (>10), slow precipitation of calcium (Ca) and/or magnesium (Mg) carbonates leads to clogging of the ICP-MS nebulizer. We propose preventing this precipitation by adding the chelating agent ethylenediaminetetraacetic acid (EDTA) at 0.1% to a 2% ammonium hydroxide matrix. This concentration of EDTA sufficed for most drinking water samples studied, as long as a 1:1 molar ratio of EDTA to Ca+Mg concentration in the water was maintained. The limit of quantitation of the developed method for iodine was < 0.1 µg L−1. The average iodine concentration in various brands of bottled mineral water sold in Israel was relatively low (average value of seven brands ± standard deviation was 7.67 ± 6.38 µg I L−1). . Regular consumption of either desalinated water or bottled mineral water probably does not supply enough iodine to eliminate iodine deficiency in Israeli consumers. Therefore, continuous follow-up of the iodine status in both tap and bottled water is strongly recommended.
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•A method for iodine determination in water samples in the alkaline matrix was developed.•Method limit of quantitation reached was < 0.1 µg L−1.•Elevating pH of mineral water led to carbonates precipitation and nebulizer clogging.•The problem of clogging was solved by EDTA (0.1%) addition to the matrix.•Iodine concentration in bottled water brands studied was found to be low.
Water desalination has been extensively developed in Israel, particularly in the last decade. The desalination process provides fresh water that typically lacks minerals, and among these are ions ...that are essential to human health and/or to agricultural production, such as Mg. We analyzed 28 tap water samples originating from different cities across Israel to document their concentrations of Mg and other elements. The data from this survey (summer 2016) were compared with the results of similar observations conducted in 2008. Regarding toxic elements, tap water across Israel does not pose any health risk for consumers and may be used as drinking water without any household pretreatment. This condition has not changed since 2008. However, the problem of Mg deficiency due to the use of desalinated water was observed in about half of the sampling locations in 2016, whereas no Mg deficiency had been detected in 2008. Moreover, household filtration of tap water prior to consumption as drinking water may worsen the situation due to the Mg status resulting from rejection of this ion; this could be harmful to the consumer, particularly under prolonged exposure.
•Tap water in Israel is of good quality and potable.•Use of desalinated water in Israel leads to Mg deficiency in drinking water.•Household filtration of tap water worsens Mg deficiency.•Tap water sources are: ground water, surface water and desalinated water.•Strong daily changes in mineral content of tap water due to mixing of water sources.
Abstract
Background
HIV persists in a long-lived infected CD4 T cell reservoir, which harbors an integrated transcriptionally latent virus. We reported that cytotoxic CD8 T cells conjugate with, and ...kill, autologous HIV-infected CD4 T cells isolated from all stages of HIV infection including reservoir cells of patients under antiretroviral therapy (ART) (image 1). This killing is attenuated by HIV Nef protein. We also previously reported that FasL presented by the cytotoxic T cells interacts with Fas on the surface of the target cells, resulting in the apoptosis of the target cells. Wajant demonstrated that Fas/FasL interaction can result in the target cell activation through NFkB, whereas NFkB binding site is present in the Long Terminal Repeat of HIV. Therefore, we hypothesize that the interaction of FasL and autologous CD8 T cells with CD4 T cells can result in the reactivation of latent HIV.
Conjugation and apoptosis of CD4 T cells by autologous CD8 T cells procured from patients on ART and analyzed by ImageStream.
The CD4 T cells in apoptosis are positive for activated caspase 3 (yellow) and DNA fragmentation/ TUNEL assay (orange). CD4 T cells (green), CD8 T cells (red), DAPI (blue).
Methods
CD4 T cells and CD8 T cells were procured from the PBMC of 20 HIV-infected patients on ART with undetectable viral load and 10 healthy volunteers. The cells were isolated using magnetic beads. Resting memory CD4 T cells (CD25-, CD69- and HLA-DR-) were isolated using a two-step bead depletion purification procedure. These cells were then incubated with soluble FasL (sFasL) and autologous CD8 T cells, for 2 and 16 hours. P24 was looked for in the supernatant by ELISA, and the expression of viral proteins was looked for inside CD4 T cells using immunohistochemistry and confocal microscopy
Results
CD4 T cells and resting memory CD4 T cells, procured from PBMC of HIV-infected patients on ART, showed HIV reactivation after incubation with sFasL and autologous CD8 T cells. This reactivation was demonstrated by the appearance of P24 in the supernatant (figure 1). HIV proteins p24, gp120, and Nef appeared inside the CD4 T cells (Image 2). HIV reactivation was not demonstrated in the CD4 T cells procured from HIV-infected patients that were incubated without sFasL or autologous CD8 T cells for 18 hours (figure 1).
Immunohistochemistry and confocal microscopy of CD4 T cells incubated with sFasL for 18 hours.
The cells were then incubated with a primary fluorescent antibody against HIV proteins (P24, Nef, GP120) and a secondary antibody ALEXA594 (red). The cells were also marked with DAPI (blue). We can see the expression of HIV proteins inside the CD4 T cells (red arrows).
ELISA for the expression of P24 in the supernatant of CD4 T cells procured from HIV-infected patients, following different manipulations
On the left, we note positive ELISA for P24 in the supernatant of the CD4 T cells of patients 11 to 16 (pg/ml). On the right, we note positive ELISA for P24 in the supernatant of the latent CD4 T cells of patients 17-20 (pg/ml).
Conclusion
HIV manipulates the cellular immune system in two ways; First, HIV attenuates CD8 T cells killing of HIV-infected CD4 T cells by the HIV-Nef protein. Second, reactivation of latent HIV by CD8 T cells and sFasL, that results in further infection of additional CD4 T cells.
Disclosures
All Authors: No reported disclosures
Teflon-coated magnetic stir bars are suspected to be a source of impurities for the laboratory blank solutions. To evaluate the ability of magnetic bars to absorb and release different elements at ...sub-ppb and above-ppb (µg L
−1
) level, the experiment with new and used bars was conducted. The bars were subjected to microwave-assisted acid digestion with the soil sample and a series of successive cleaning procedures. The spectrometry analysis of the obtained extracts revealed that most of the elements may be released during subsequent extractions in the ppb-level concentrations, whereas the following elements: Cr, Cu, Sb, Sn, and Pb may be extracted even in elevated concentrations. Electronic microscope inspection of Teflon surfaces demonstrated the multiple defects that probably increase the absorption of the elements. We concluded that the concentration of mineral impurities in the laboratory blank solution prepared in the randomly selected one vessel per sample preparation batch is hardly propagated to the other used vessels. The alternative concept "blank per vessel" was proposed especially for ultra-trace analysis. Also, an additional step in the bar washing procedure was proposed.
Graphical abstract
Summary
Peripheral blood mononuclear cells (
PBMC
) of untreated,
HIV
‐infected patients contain
HIV
‐specific
CD
8 T cells as well as their corresponding targets,
HIV
‐infected
CD
4 T cells. To ...determine if
CD
4 T‐cell depletion in
HIV
‐infected patients may result from autologous
CD
8–
CD
4 T‐cell interaction,
CD
8 and
CD
4 T cells procured from
PBMC
of acute and chronic untreated
HIV
‐infected patients were sorted and co‐incubated. Formation of
CD
8‐
CD
4 T‐cell conjugates was observed by fluorescence microscopy. Apoptosis of
CD
4 T cells in conjugation was recorded by digitized images and was further observed and measured by
FACS
using Annexin staining. Perforin expression in the
CD
8 T cells was measured using intracellular monoclonal perforin antibody staining.
HIV DNA
in the conjugated
CD
4 T cells was detected by
in situ
PCR
. We found that 6·1 ± 0·5% of
CD
4 T cells from acute
HIV
‐infected patients and 3·0 ± 0·5% from chronic
HIV
‐infected patients formed
CD
8–
CD
4 T‐cell conjugates. Annexin binding and cell morphology typical of apoptosis were observed in the conjugated
CD
4 T cells. The majority of
CD
8 T cells that had conjugated to
CD
4 T cells expressed perforin. The conjugated
CD
4 T cells exhibited nuclear
HIV DNA
.
CD
8 T cells and
HIV
‐infected
CD
4 T cells, both procured from the
PBMC
of untreated
HIV
‐infected patients, form conjugates. Apoptotic lytic activity has been observed in the conjugated
CD
4 T cells. We propose that
CD
4 T‐cell annihilation in
HIV
‐infected patients results, at least in part, from the interactions of perforin‐rich
CD
8 T cells with autologous,
HIV
‐infected
CD
4 T cells.
Although CD8⁺ cytotoxic T lymphocytes (CTL) exhibit both Fas ligand (FasL) -based and perforin-based lytic activities, the accepted hallmark of a fully active CTL remains its perforin killing ...machinery. Yet the origin, rationale for possessing both a slow-acting (FasL) and a fast-acting (perforin) killing mechanism has remained enigmatic. Here we have investigated perforin expression in CTL directly involved in acute tumour (i.e. leukaemias EL4 and L1210) allograft rejection occurring within the peritoneal cavity. We show that at the height of the immune response, the majority of conjugate-forming CD8⁺ CTL express high levels of perforin messenger RNA and protein, and kill essentially via perforin. Later however, coinciding with complete rejection, fully cytocidal CTL emerge which exhibit a stark decrease in perforin and now kill preferentially via constitutively expressed FasL. Although late in emergence, and persistent, these powerful CTL are neither effector-memory nor memory CTL. This finding has implications for the monitoring of anti-transplant responses in clinical settings, based on assessing perforin expression in graft infiltrating CD8⁺ T cells. The results show that as the immune response progresses in vivo, targeted cellular suicide mainly prunes high perforin-expressing CD8⁺ cells, resulting in the gradual switch in effector CTL, from mostly perforin-based to largely Fas/FasL-based killers. Hence, two kinds of CD8⁺ CTL have two killing strategies.
A 970 bp cDNA Na+/glucose cotransporter (SGLT1) was isolated and sequenced from chicken jejunum by reverse transcriptase polymerase chain reaction (RT-PCR) using primers based on conserved regions. ...Using the 970 bp PCR product as a specific probe, Northern Blot hybridization indicated a transcript of ca. 4 kb. The isolated chicken intestinal SGLT1 cDNA was used to quantitate mRNA expression. Glucose uptake activity and kinetics were determined in brush border membrane vesicles (BBMV) from jejunum tissue of chickens which were either fed, food-deprived or refed following food deprivation. Net glucose uptake to BBMV was higher (P < 0.02) in the control and refed chicks (149 ± 11.9, 139.6 ± 7.43 pmol · mg protein−1 · s−1) than in food-deprived chicks (107 ± 4.23 pmol · mg protein−1 · s−1). The km (150 μmol/L) and Vmax (1111.1 pmol · mg protein−1 · s−1) were higher in the food-deprived chicks compared to control and refed birds (25, 24 μmol/L and 227, 142 pmol · mg protein−1 · s−1, respectively). Expression of SGLT1 mRNA was significantly enhanced in the food-deprived and refed birds. In food-deprived chicks the lower affinity and higher activity of the SGLT1 transporter for glucose were accompanied by higher expression of mRNA which might indicate that the transporter was upregulated by low substrate concentration. Quantification of expression of intestinal mRNA of SGLT1 provides important information concerning control of nutrient uptake. J. Nutr. 130: 2174–2179, 2000.
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