Use of pediatric thymus to humanize mice for HIV-1 mucosal transmission.

Humanized mice have been used to study human immunodeficiency virus type 1 (HIV-1) transmission, pathogenesis, and treatment. The ability of pediatric thymus tissue implanted either in the leg (Leg PedThy) or under the renal capsule (Renal PedThy) with allogeneic CD34+ hematopoietic cells (HSCs) in NSG mice was evaluated for reconstitution of human immune cells and for rectal transmission of HIV-1. These mice were compared to traditional BLT mice implanted with fetal liver and thymus under the renal capsule and mice injected only with HSCs. Renal PedThy mice had similar immune reconstitution in the blood, spleen and intestine as BLT mice, while Leg PedThy mice had transient detection of immune cells, particularly CD4+ T cells and macrophages, the target cells for HIV-1 infection. Rectal transmission and replication of HIV-1 was efficient in BLT mice but lower and more variable in Renal PedThy mice. HIV-1 was poorly transmitted in HSC mice and not transmitted in Leg PedThy mice, which correlated with the frequencies of target cells in the spleen and intestine. Humanization of NSG mice with pediatric thymus was successful when implanted under the kidney capsule, but led to less efficient HIV-1 rectal transmission and replication compared to BLT mice.

Targeting spike glycans to inhibit SARS-CoV2 viral entry.

SARS-CoV-2 spike harbors glycans which function as ligands for lectins. Therefore, it should be possible to exploit lectins to target SARS-CoV-2 and inhibit cellular entry by binding glycans on the spike protein. Burkholderia oklahomensis agglutinin (BOA) is an antiviral lectin that interacts with viral glycoproteins via N-linked high mannose glycans. Here, we show that BOA binds to the spike protein and is a potent inhibitor of SARS-CoV-2 viral entry at nanomolar concentrations. Using a variety of biophysical approaches, we demonstrate that the interaction is avidity driven and that BOA cross-links the spike protein into soluble aggregates. Furthermore, using virus neutralization assays, we demonstrate that BOA effectively inhibits all tested variants of concern as well as SARS-CoV 2003, establishing that multivalent glycan-targeting molecules have the potential to act as pan-coronavirus inhibitors.

Targeting Spike Glycans to Inhibit SARS-CoV2 Viral Entry.

SARS-CoV-2 Spike harbors glycans which function as ligands for lectins. Therefore, it should be possible to exploit lectins to target SARS-CoV-2 and inhibit cellular entry by binding glycans on the Spike protein. Burkholderia oklahomensis agglutinin (BOA) is an antiviral lectin that interacts with viral glycoproteins via N-linked high mannose glycans. Here, we show that BOA binds to the Spike protein and is a potent inhibitor of SARS-CoV-2 viral entry at nanomolar concentrations. Using a variety of biophysical tools, we demonstrate that the interaction is avidity driven and that BOA crosslinks the Spike protein into soluble aggregates. Furthermore, using virus neutralization assays, we demonstrate that BOA effectively inhibits all tested variants of concern as well as SARS-CoV 2003, establishing that glycan-targeting molecules have the potential to be pan-coronavirus inhibitors.

CG Dinucleotide Removal in Bioluminescent and Fluorescent Reporters Improves HIV-1 Replication and Reporter Gene Expression for Dual Imaging in Humanized Mice.

Visualizing the transmission and dissemination of human immunodeficiency virus type 1 (HIV-1) in real time in humanized mouse models is a robust tool to investigate viral replication during treatments and in tissue reservoirs. However, the stability and expression of HIV-1 reporter genes are obstacles for long-term serial imaging in vivo. Two replication-competent CCR5-tropic HIV-1 reporter constructs were created that encode either nanoluciferase (nLuc) or a near-infrared fluorescent protein (iRFP) upstream of nef. HIV-1 reporter virus replication and reporter gene expression was measured in cell culture and in humanized mice. While reporter gene expression in vivo correlated initially with plasma viremia, expression decreased after 4 to 5 weeks despite high plasma viremia. The reporter genes were codon optimized to remove cytosine/guanine (CG) dinucleotides, and new CO-nLuc and CO-iRFP viruses were reconstructed. Removal of CG dinucleotides in HIV-1 reporter viruses improved replication in vitro and reporter expression in vivo and ex vivo. Both codon-optimized reporter viruses could be visualized during coinfection and in vivo reporter gene expression during treatment failure preceded detection of plasma viremia. While the dynamic range of CO-iRFP HIV-1 was lower than that of CO-nLuc HIV-1, both viruses could have utility in studying and visualizing HIV-1 infection in humanized mice. IMPORTANCE Animal models are important for studying HIV-1 pathogenesis and treatments. We developed two viruses each encoding a reporter gene that can be expressed in cells after infection. This study shows that HIV-1 infection can be visualized by noninvasive, whole-body imaging in mice with human immune cells over time by reporter expression. We improved reporter expression to reflect HIV-1 replication and showed that two viral variants can be tracked over time in the same animal and can predict failure of antiretroviral therapy to suppress virus.

Long-Acting Rilpivirine (RPV) Preexposure Prophylaxis Does Not Inhibit Vaginal Transmission of RPV-Resistant HIV-1 or Select for High-Frequency Drug Resistance in Humanized Mice.

As a long-acting formulation of the nonnucleoside reverse transcriptase inhibitor rilpivirine (RPV LA) has been proposed for use as preexposure prophylaxis (PrEP) and the prevalence of transmitted RPV-resistant viruses can be relatively high, we evaluated the efficacy of RPV LA to inhibit vaginal transmission of RPV-resistant HIV-1 in humanized mice. Vaginal challenges of wild-type (WT), Y181C, and Y181V HIV-1 were performed in mice left untreated or after RPV PrEP. Plasma viremia was measured for 7 to 10 weeks, and single-genome sequencing was performed on plasma HIV-1 RNA in mice infected during PrEP. RPV LA significantly prevented vaginal transmission of WT HIV-1 and Y181C HIV-1, which is 3-fold resistant to RPV. However, it did not prevent transmission of Y181V HIV-1, which has 30-fold RPV resistance in the viruses used for this study. RPV LA did delay WT HIV-1 dissemination in infected animals until genital and plasma RPV concentrations waned. Animals that became infected despite RPV LA PrEP did not acquire new RPV-resistant mutations above frequencies in untreated mice or untreated people living with HIV-1, and the mutations detected conferred low-level resistance. These data suggest that high, sustained concentrations of RPV were required to inhibit vaginal transmission of HIV-1 with little or no resistance to RPV but could not inhibit virus with high resistance. HIV-1 did not develop high-level or high-frequency RPV resistance in the majority of mice infected after RPV LA treatment. However, the impact of low-frequency RPV resistance on virologic outcome during subsequent antiretroviral therapy still is unclear.IMPORTANCE The antiretroviral drug rilpivirine was developed into a long-acting formulation (RPV LA) to improve adherence for preexposure prophylaxis (PrEP) to prevent HIV-1 transmission. A concern is that RPV LA will not inhibit transmission of drug-resistant HIV-1 and may select for drug-resistant virus. In female humanized mice, we found that RPV LA inhibited vaginal transmission of WT or 3-fold RPV-resistant HIV-1 but not virus with 30-fold RPV resistance. In animals that became infected despite RPV LA PrEP, WT HIV-1 dissemination was delayed until genital and plasma RPV concentrations waned. RPV resistance was detected at similar low frequencies in untreated and PrEP-treated mice that became infected. These results indicate the importance of maintaining RPV at a sustained threshold after virus exposure to prevent dissemination of HIV-1 after vaginal infection and low-frequency resistance mutations conferred low-level resistance, suggesting that RPV resistance is difficult to develop after HIV-1 infection during RPV LA PrEP.

Differential Activation of the Transcription Factor IRF1 Underlies the Distinct Immune Responses Elicited by Type I and Type III Interferons.

Type I and III interferons (IFNs) activate similar downstream signaling cascades, but unlike type I IFNs, type III IFNs (IFNλ) do not elicit strong inflammatory responses in vivo. Here, we examined the molecular mechanisms underlying this disparity. Type I and III IFNs displayed kinetic differences in expression of IFN-stimulated genes and proinflammatory responses, with type I IFNs preferentially stimulating expression of the transcription factor IRF1. Type III IFNs failed to induce IRF1 expression because of low IFNλ receptor abundance and insufficient STAT1 activation on epithelial cells and thus did not activate the IRF1 proinflammatory gene program. Rather, IFNλ stimulation preferentially induced factors implicated in tissue repair. Our findings suggest that IFN receptor compartmentalization and abundance confer a spatiotemporal division of labor where type III IFNs control viral spread at the site of the infection while restricting tissue damage; the transient induction of inflammatory responses by type I IFNs recruits immune effectors to promote protective immunity.

Predictors of bacteremia in infants with diarrhea and systemic inflammatory response syndrome attending an urban diarrheal treatment center in a developing country.

OBJECTIVES: To identify clinical and laboratory predictors of bacteremia in infants with diarrhea and systemic inflammatory response syndrome and to analyze their outcome. STUDY DESIGN: Retrospective, case-control study. SETTING: The Special Care Ward of the Dhaka Hospital of the International Centre for Diarrheal Disease Research, Bangladesh, Dhaka, Bangladesh. PATIENTS: All the infants (n = 90) admitted to the Special Care Ward between May 2005 and April 2006 who had a blood culture, full peripheral blood count, and serum C-reactive protein performed were included in the study. Infant with systemic inflammatory response syndrome with confirmed bacteremia (n = 18) constituted cases, and those with systemic inflammatory response syndrome but negative blood culture (n = 72) constituted the controls. RESULTS: The following features were analyzed by comparing the two groups: absent or uncountable peripheral pulses, hypothermia, sclerema, altered mental status, white blood cell count, serum C-reactive protein, total protein concentrations, and outcome. The case-fatality rate was significantly higher among bacteremic infants compared with those without bacteremia (33% vs. 6%, p < .01). In the univariate model, sclerema (56% vs. 28%, p = .05), hyperglycemia (28% vs. 6%, p < .01), immature neutrophils [3.5 (00, 6.5) vs. 0.0 (0.0, 3.25); p = .02], higher C-reactive protein [2.7 (1.2, 7.4) vs. 1.8 (0.5, 4.2); p = .02], and lower serum total protein (51.1 +/- 14.1 vs. 57.6 +/- 12.2; p = .05) were identified as potential predictors of bacteremia. However, in the logistic regression analysis, after adjusting for confounders, only hypothermia (odds ratio = 6.4, 95% confidence Interval, 1.6-25.9; p = .01) and absent or uncountable peripheral pulse (odds ratio, 12.4, 95% confidence interval, 1.9-83.4; p < .01) remained significant independent predictors of bacteremia. CONCLUSIONS: Our data suggest that, in infants presenting with diarrhea and systemic inflammatory response syndrome, coexistence of hypothermia and absent or uncountable peripheral pulse is strongly associated with bacteremia. Bacteremia in this patient group is associated with high case-fatality rates.

Buffer Standards for the Physiological pH of the Zwitterionic Compound, DIPSO from 5 to 55 degrees C.

The values of the second dissociation constant, pK(2), and related thermodynamic quantities of 3-[N,N-bis (2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO) have already been reported over the temperature range 5 to 55 degrees C including 37 degrees C. This paper reports the pH values of four NaCl-free buffer solutions and four buffer composition containing NaCl salt at I = 0.16 mol.kg(-1). Conventional pa(H) values are reported for all eight buffer solutions. The operational pH values have been calculated for four buffer solutions recommended as pH standards, at 25 and 37 degrees C after correcting the liquid junction potentials with the flowing junction cell.

Buffer Standards for the Biochemical pH of 3-(N-morpholino)-2-hydroxypropanesulfonic Acid from (278.15 to 328.15) K.

The values of the second dissociation constant pK(2) and related thermodynamic quantities of the ampholyte 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO) have been previously determined at temperatures from (278.15 to 328.15) K. In this study, the pH values of two buffer solutions without NaCl and three buffer solutions with NaCl having ionic strengths (I = 0.16 mol·kg(-1)) similar to those in blood plasma, have been evaluated at 12 temperatures from (278.15 to 328.15) K using an extended form of the Debye-Hückel equation, since the Bates-Guggenheim convention is valid up to I = 0.1 mol·kg(-1). The liquid junction potentials (E(j)) between the buffer solutions of MOPSO and saturated KCl solution of the calomel electrode at (298.15 and 310.15) K have been estimated by measurement with a flowing junction cell. These values of E(j) have been used to ascertain the operational pH values at (298.15 and 310.15) K. Three buffer solutions of MOPSO are recommended as useful reference solutions for pH measurements in saline media of ionic strength I = 0.16 mol·kg(-1).

Buffer Standards for the Physiological pH of the Zwitterionic Compound, ACES from 5 to 55°C.

The values of the second dissociation constant pK2 and related thermodynamic quantities of [N-(2-acetamido)-2-aminoethanesulfonic acid] (ACES) have already been reported over the temperature range 5 to 55°C including 37°C. This paper reports the paH values of four chloride ion free buffer solutions and eight buffer solutions with I = 0.16 mol·kg -1, matching closely to that of the physiological sample. Conventional paH values for all twelve buffer solutions from 5 to 55°C, are reported. The residual liquid junction potential correction for two widely used temperatures, 25 and 37°C, has been made. The flowing-junction calomel cell method has been utilized to measure Ej , the liquid junction potential. The operational pH values for four buffer solutions at 25 and 37°C are calculated using the physiological phosphate buffer standard based on NBS/NIST convention. These solutions are recommended as pH standards in the pH range of 6.8 to 7.2 for physiological fluids.

Buffer Standards for pH Measurement of N-(2-Hydroxyethyl)piperazine-N'-2-ethanesulfonic Acid (HEPES) for I = 0.16 mol.kg from 5 to 55 degrees C.

The values of the second dissociation constant, pK(2) of N-(2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid (HEPES) have been reported at 12 temperatures over the temperature range 5 to 55 degrees C, including 37 degrees C. This paper reports the results for the pa(H) of eight isotonic saline buffer solutions with an I = 0.16 mol*kg(-1) including compositions: (a) HEPES (0.01 mol*kg(-1)) + NaHEPES (0.01 mol*kg(-1)) + NaCl (0.15 mol*kg(-1)); (b) HEPES (0.02 mol*kg(-1)) + NaHEPES (0.02 mol*kg(-1)) + NaCl (0.14 mol*kg(-1)); (c) HEPES (0.03 mol*kg(-1)) + NaHEPES (0.03 mol*kg(-1)) + NaCl (0.13 mol*kg(-1)); (d) HEPES (0.04 mol*kg(-1)) + NaHEPES (0.04 mol*kg(-1)) + NaCl (0.12 mol*kg(-1)); (e) HEPES (0.05 mol*kg(-1)) + NaHEPES (0.05 mol*kg(-1)) + NaCl (0.11 mol*kg(-1)); (f) HEPES (0.06 mol*kg(-1)) + NaHEPES (0.06 mol*kg(-1)) + NaCl (0.10 mol*kg(-1)); (g) HEPES (0.07 mol*kg(-1)) + NaHEPES (0.07 mol*kg(-1)) + NaCl (0.09 mol*kg(-1)); and (h) HEPES (0.08 mol*kg(-1)) + NaHEPES (0.08 mol*kg(-1)) + NaCl (0.08 mol*kg(-1)). Conventional pa(H) values, for all eight buffer solutions from 5 to 55 degrees C have been calculated. The operational pH values with liquid junction corrections, at 25 and 37 degrees C have been determined based on the NBS/NIST standard between the physiological phosphate standard and four buffer solutions. These are recommended as pH standards for physiological fluids in the range of pH 7.3 to 7.5 at I = 0.16 mol*kg(-1).

Anthropometric characteristics of elderly people: observations at a large diarrheal hospital in Dhaka, Bangladesh.

There is a lack of evidence-based information to assist health policy makers in preparing for appropriate health, nutrition, and social-support guidelines for the elderly in Bangladesh. We examined selected indicators of the nutritional status of elderly people attending the Dhaka Hospital of ICDDR,B, Dhaka, Bangladesh. The population constituted of 1,196 individuals (718 men and 478 women), aged 60 to 106 years, who attended the hospital between 1 January 1993 and 31 December 2003. Patients were recruited from a hospital-based systematic sampling, regardless of age and gender, that presented to the facility. Men were heavier, and taller than women were (p < 0.001 for both comparisons). Using MUAC cut-off of < 22 cm for females and < 23 cm for males, at least 50% of the elderly were peripherally wasted (malnourished). Among all the study population, 40% had a BMI within the optimal range (18.5-24.9 kg/m(2)). Using the chronic energy deficiency (CED) classification, at least half of elderly (> or= 60 year) women were chronic energy deficient (BMI < 18.5). A significantly higher proportion of elderly women (7%) compared to men (2%) were overweight (BMI > or = 25, p < 0.001). Among the elderly ( > or = 60 year), males and females from a higher socioeconomic status (SES) had significantly higher BMI (p < 0.001, p = 0.001, respectively) and MUAC values (p < 0.001, p < 0.001, respectively) than their less well-off SES counterparts. We consider that, although our data were not valid for assessing the country situation, they are still useful as baseline information for longitudinal studies and for highlighting the need for studies in other geographical locations and in other population groups.