TNF and CD28 Signaling Play Unique but Complementary Roles in the Systemic Recruitment of Innate Immune Cells after Staphylococcus aureus Enterotoxin A Inhalation.

Staphylococcus aureus enterotoxins cause debilitating systemic inflammatory responses, but how they spread systemically and trigger inflammatory cascade is unclear. In this study, we showed in mice that after inhalation, Staphylococcus aureus enterotoxin A rapidly entered the bloodstream and induced T cells to orchestrate systemic recruitment of inflammatory monocytes and neutrophils. To study the mechanism used by specific T cells that mediate this process, a systems approach revealed inducible and noninducible pathways as potential targets. It was found that TNF caused neutrophil entry into the peripheral blood, whereas CD28 signaling, but not TNF, was needed for chemotaxis of inflammatory monocytes into blood and lymphoid tissue. However, both pathways triggered local recruitment of neutrophils into lymph nodes. Thus, our findings revealed a dual mechanism of monocyte and neutrophil recruitment by T cells relying on overlapping and nonoverlapping roles for the noninducible costimulatory receptor CD28 and the inflammatory cytokine TNF. During sepsis, there might be clinical value in inhibiting CD28 signaling to decrease T cell-mediated inflammation and recruitment of innate cells while retaining bioactive TNF to foster neutrophil circulation.

Recombinant immunotoxin engineered for low immunogenicity and antigenicity by identifying and silencing human B-cell epitopes.

Recombinant immunotoxins (RITs) are hybrid proteins used to treat cancer. These proteins are composed of an Fv that reacts with cancer cells joined to a portion of Pseudomonas exotoxin A, which kills the cell. Because the toxin is a foreign protein, it can induce neutralizing antibodies and thereby limit the number of doses a patient can receive. We previously identified seven major mouse B-cell epitopes in the toxin, and subsequently silenced them using point mutations that converted large hydrophilic amino acids to alanine, yet retained full antitumor activity. Here we present results in which we identify and silence human B-cell epitopes in the RIT HA22. We obtained B cells from patients with antibodies to RITs, isolated the corresponding variable fragments (Fvs), and constructed a phage-display library containing Fvs that bind to the RITs. We then used alanine scanning mutagenesis to locate the epitopes. We found that human and mouse epitopes frequently overlap but are not identical. Most mutations that remove mouse epitopes did not remove human epitopes. Using the epitope information, we constructed a variant immunotoxin, HA22-LR-LO10, which has low reactivity with human antisera, yet has high cytotoxic and antitumor activity and can be given to mice at high doses without excess toxicity. The toxin portion of this RIT (LR-LO10) can be used with Fvs targeting other cancer antigens and is suitable for clinical development.

Association between metal implants and urinary chromium levels in US adults: a cross-sectional study from NHANES.

Metal implants play a significant role in orthopedics, commonly used for treating fractures, joint replacement surgeries, spinal procedures, and more. Chromium (Cr), crucial in these implants, may raises health concerns. However, the relationship between metal implants and urine Cr levels remains uncertain. We aimed to evaluate this relationship. We conducted a cross-sectional study on 1419 individuals aged 40 years or older using data from the National Health and Nutrition Examination Survey (NHANES) spanning the years 2017 to 2020. Multivariate linear regression models and subgroup analysis were applied to assess associations between metal implants and urine Cr levels. Among the 1419 participants, 402 [28.3%] self-reported having metal objects in their bodies. After adjusting for potential confounding factors, metal implants were positively correlated with the accumulation of urine Cr (ß = 0.41, 95% CI 0.04-0.77, p = 0.028). However, the positive correlation of metal implants with urine Cr was only present in females (ß = 0.81, 95% CI 0.08-1.53, p = 0.029), but not in males. Our study revealed higher urine Cr levels in individuals with metal implants, with noticeable gender differences. Additionally, those with metal implants exhibited a more pronounced elevation in urine Cr levels with increasing age compared to individuals without implants.

The Impact of Contrast-Associated Acute Kidney Injury on All-Cause Mortality in Older Patients After Coronary Angiography:A 7.5-year Follow-Up.

Older patients (≥75 years) after coronary angiography constitute an increasing proportion, but only limited data are available regarding the prognosis of geriatric contrast-associated acute kidney injury (CA-AKI). Patients (≥75 years) undergoing coronary angiography between December 2010 and September 2013 were consecutively enrolled. CA-AKI was defined as an increase in serum creatinine of 25% or .5 mg/dL from the baseline within 48-72 h of contrast exposure. All-cause mortality was assessed during median 7.5 years (interquartile range [IQR] 6.7-8.7 years) follow-up period. In total, 571 patients aged >75 years undergoing coronary angiography were enrolled in a single center study; 82 (14.4%) patients had CA-AKI. The all-cause mortality during the median 7.5 years follow-up period was 22.0% in patients with CA-AKI and 13.1% in patients without CA-AKI (P = .015). After adjusting for potential confounding factors, the multivariable analysis indicated that CA-AKI was related to an increased risk of all-cause mortality during the median 7.5-year follow-up (hazard ratio [HR]: 2.46; 95% CI: 1.29-4.7; P = .006). CA-AKI is a significant and independent predictor of long-term mortality for patients aged over 75 years who underwent coronary angiography.

Improving pedicle screw path planning by vertebral posture estimation.

Objective.Robot-assisted pedicle screw placement in spinal surgery can reduce the complications associated with the screw placement and reduce the hospital return counts due to malfunctions. However, it requires accurate planning for a high-quality procedure. The state-of-the-art technologies reported in the literature either ignore the anatomical variations across vertebrae or require substantial human interactions. We present an improved approach that achieves pedicle screw path planning through multiple projections of a numerically re-oriented vertebra with the estimated posture.Approach.We proposed an improved YOLO-type neural network model (YOLOPOSE3D) to estimate the posture of a vertebra before pedicle path planning. In YOLOPOSE3D, the vertebral posture is given as a rotation quaternion and 3D location coordinates by optimizing the intersection over union of the vertebra with the predicted posture and the actual posture. Then, a new local coordinate system is established for the vertebra based on the estimated posture. Finally, the optimal pedicle screw path trajectory is determined from the multiple projections of the vertebra in the local coordinates.Main results.The experimental results in difficult cases of scoliosis showed that the new YOLOPOSE3D network could accurately detect the location and posture of the vertebra with average translation and orientation errors as small as 1.55 mm and 2.55°. The screw path planning achieved 83.1% success rate without breaking the pedicle cortex for the lumbar vertebral L1-L5, which is better than that of a doctor's manual planning, 82.4%. With the clinical class A requirement to allow less than 2 mm out of the pedicle cortex, the success rate achieved nearly 100%.Significance.The proposed YOLOPOSED3D method can accurately determine the vertebral postures. With the improved posture prior, better clinical outcomes can be achieved for pedicle screw placement in spine internal fixation procedures.

Robust production of monovalent bispecific IgG antibodies through novel electrostatic steering mutations at the CH1-Cλ interface.

Bispecific antibodies represent an increasingly large fraction of biologics in therapeutic development due to their expanded scope in functional capabilities. Asymmetric monovalent bispecific IgGs (bsIgGs) have the additional advantage of maintaining a native antibody-like structure, which can provide favorable pharmacology and pharmacokinetic profiles. The production of correctly assembled asymmetric monovalent bsIgGs, however, is a complex engineering endeavor due to the propensity for non-cognate heavy and light chains to mis-pair. Previously, we introduced the DuetMab platform as a general solution for the production of bsIgGs, which utilizes an engineered interchain disulfide bond in one of the CH1-CL domains to promote orthogonal chain pairing between heavy and light chains. While highly effective in promoting cognate heavy and light chain pairing, residual chain mispairing could be detected for specific combinations of Fv pairs. Here, we present enhancements to the DuetMab design that improve chain pairing and production through the introduction of novel electrostatic steering mutations at the CH1-CL interface with lambda light chains (CH1-Cλ). These mutations work together with previously established charge-pair mutations at the CH1-CL interface with kappa light chains (CH1-Cκ) and Fab disulfide engineering to promote cognate heavy and light chain pairing and enable the reliable production of bsIgGs. Importantly, these enhanced DuetMabs do not require engineering of the variable domains and are robust when applied to a panel of bsIgGs with diverse Fv sequences. We present a comprehensive biochemical, biophysical, and functional characterization of the resulting DuetMabs to demonstrate compatibility with industrial production benchmarks. Overall, this enhanced DuetMab platform substantially streamlines process development of these disruptive biotherapeutics.

Condensation Separation of NO2 with Dimerization Reaction in the Presence of Noncondensable Gas: Critical Assessment and Model Development.

Pure nitrogen dioxide (NO2) has significant economic value and is widely used in many fields, for which condensation technology plays an important role in separation and purification. However, developing cost-effective NO2 condensers remains challenging due to the lack of precise theoretical guidelines and comprehensive understanding of NO2 condensation process. In this work, NO2 condensation at various inlet surface subcoolings, mole fractions of noncondensable gas (NCG), and Re numbers was studied with a visualization experimental system. The influential rules of each parameter on heat transfer coefficients (HTCs) and the NO2 condensate state as the coexistence of droplet, streamlet and film were revealed. A substantial underestimation of experimental data by the classical heat and mass transfer analogy (HMTA) model was quantified. The large discrepancy was found to originate from the uniqueness in heat transfer, mass transfer, and condensate state caused by NO2 dimerization during condensation. A modified HMTA model was developed considering the release heat of dimerization reaction and the promotion of mass transfer by an increased NO2 concentration gradient within the diffusion layer which contribute to improvements of HTCs by ∼6 and ∼49%, respectively. The correction of liquid film roughness regarding potential heterogeneity of dimerization was proposed as a function of the key parameters, contributing to the improvement of HTCs by ∼150%. An accurate theoretical formula for HTCs prediction within an error of ±25% was finally derived, providing the key step for success in practical applications.

An improved lentiviral system for efficient expression and purification of ß-defensins in mammalian cells.

ß-Defensins are a family of conserved small cationic antimicrobial peptides with different significant biological functions. The majority of mammalian ß-defensins are expressed in epididymis, and many of them are predicted to have post-translational modifications. However, only a few of its members have been well studied due to the limitations of expressing and purifying bioactive proteins with correct post-translational modifications efficiently. Here we developed a novel Fc tagged lentiviral system and Fc tagged prokaryotic expression systems provided new options for ß-defensins expression and purification. The novel lentiviral system contains a secretive signal peptide, an N-terminal IgG Fc tag, a green fluorescent protein (GFP), and a puromycin selection marker to facilitate efficient expression and fast purification of ß-defensins by protein A magnetic or agarose beads. It also enables stable and large-scale expression of ß-defensins with regular biological activities and post-translational modification. Purified ß-defensins such as Bin1b and a novel human ß-defensin hBD129 showed antimicrobial activity, immuno-regulatory activity, and expected post-translational phosphorylation, which were not found in Escherichia coli (E. coli) in expressed form. Furthermore, we successfully applied the novel system to identify mBin1b interacting proteins, explaining Bin1b in a better way. These results suggest that the novel lentiviral system is a powerful approach to produce correct post-translational processed ß-defensins with bioactivities and is useful to identify their interacting proteins. This study has laid the foundation for future studies to characterize function and mechanism of novel ß-defensins.

Construction of a fluorescein-responsive chimeric receptor with strict ligand dependency.

Since many cell functions are regulated by members of the cytokine receptor superfamily, the artificial mimicry of the cytokine receptor system would be attractive for cellular engineering. We previously showed that an antibody/cytokine receptor chimera can transduce a growth signal in response to non-natural ligands, such as fluorescein-conjugated BSA. However, considerable background of cell proliferation was observed without antigen. Therefore, we redesigned chimeric receptor constructs with different combinations of domains containing anti-fluorescein single chain Fv (ScFv), extracellular D1/D2 as well as transmembrane domains of erythropoietin receptor (EpoR), and the intracellular domain of glycoprotein 130 (gp130), to obtain strictly fluorescein-dependent chimeric receptors. When interleukin-3-dependent Ba/F3 cells were transduced with retroviral vectors encoding individual chimeric receptors, the chimeras either with both D1 and D2 domains or without any EpoR extracellular domain attained a strict ligand-dependent ON/OFF regulation.

[Non-conditional logistic regression analysis on risk factors of central venous catheter-related infection].

OBJECTIVE: To study the correlation of indwelling central venous catheter and systemic infection, to study the causative microorganisms and factors contributing to infections, and the diagnosis and treatment of such infection. METHODS: All the central venous catheters were produced by American or German companies. Subclavian vein, internal jugular vein, or femoral vein was chosen. Skin swab, blood in the catheter and peripheral blood were cultured. Clinical data including sex, age, disease, times of puncture, site of puncture, indwelling duration, type of catheter, caliber of catheter (single or double chamber, hemodialysis), nursing care, ways of use of the catheter were recorded, and they were subjected to non-conditional logistic regression analysis, then independent risk factors were determined with multivariate stepwise non-conditional logistic regression analysis. RESULTS: Unvaried non-conditional logistic regression analysis showed that sex, primary disease, site of puncture, nursing care, and ways of use of the catheter showed no correlation with CRS. Multivariate stepwise non-conditional logistic regression analysis showed that indwelling duration [odds ratio (OR)=2.682, 95% confidence interval (CI): 1.156-6.226, chi2=5.275, P=0.022], caliber of catheter (OR=2.702, 95% CI: 1.183-6.175, chi2=5.559, P=0.018) and white blood cell count (WBC)<4x10(9)/L (OR=1.371, 95% CI:1.044-1.800, chi2=5.142, P=0.023) were independent factors. The clinical study of 1,100 patients showed that the total correlated infection rate was 2.5% (28/1,100). CONCLUSION: Central venous catheterization is the major cause of bacteremia in hospital. The risk factors of infection are maintenance duration, location, and individual difference, and they are obviously correlated.

Responses to LPS boost effector CD8 T-cell accumulation outside of signals 1 and 2.

Immunization with adjuvant plus antigen induces durable T-cell immunity and is a mainstay of vaccines. Here, the consequence of separating antigen stimulation of T cells from the adjuvant response was studied in a re-transfer model. Effector CD8 T cells in recipient mice were exposed to lipopolysaccharide (LPS), the Toll-like receptor 4 (TLR4) ligand, which significantly increased persistence. While accumulation in lymphoid and non-lymphoid organs was evident, this result depended upon the timing of LPS administration and the presence of the TLR4 adaptor TRIF in the recipient mice. Interestingly, there was very little impact of the LPS response on subset differentiation, which rather appeared to be programmed by antigen and costimulation. To discern factors that limit accumulation, interleukin 10 (IL-10) was targeted since it is a product of TLR4 triggering and mitigates inflammation. Blockade of IL-10 increased accumulation even though the effector CD8 T cells were well past the priming phase, but upon recall interferon-γ secretion was not affected as would be expected when IL-10 is present during priming. Thus, the adjuvant-altered microenvironment is effective not only in the presence of antigen but also during a window of effector CD8 T-cell stasis, suggesting that pathogen-associated molecular pattern molecules released during co-infection, or by vaccines, could alter the survival fate of specific effector T cells.Cellular & Molecular Immunology advance online publication, 20 July 2015; doi:10.1038/cmi.2015.69.

Optimal CD4 T cell priming after LPS-based adjuvanticity with CD134 costimulation relies on CXCL9 production.

LPS is a powerful adjuvant, and although LPS-mediated TLR4 signaling has been exquisitely delineated, the in vivo mechanism of how TLR4 responses impact T cell priming is far less clear. Besides costimulation, TNF and type 1 IFN are dominant cytokines released after TLR4 activation and can shape T cell responses, but other downstream factors have not been examined extensively. Depending on context, we show that IFNαR1 blockade resulted in minor to major effects on specific CD4 T cell clonal expansion. To help explain these differences, it was hypothesized that IFNαR1 blockade would inhibit specific T cell migration by reducing chemokine receptor signaling, but specific CD4 T cells from IFNαR1-blocked mice were readily able to migrate in response to specific chemokines. Next, we examined downstream factors and found that type 1 IFN signaling was necessary for chemokine production, even when mice were immunized with specific Ag with LPS and CD134 costimulation. IFNαR1 signaling promoted CXCL9 and CXCL10 synthesis, suggesting that these chemokines might be involved in the LPS and CD134 costimulation response. After immunization, we show that CXCL9 blockade inhibited CD4 T cell accumulation in the liver but also in LNs, even in the presence of elevated serum IFN-ß levels. Thus, whereas type 1 IFN might have direct effects on primed CD4 T cells, the downstream chemokines that play a role during migration also impact accumulation. In sum, CXCL9 production is a key benchmark for productive CD4 T cell vaccination strategies.

Megakaryocytic potentiating factor and mature mesothelin stimulate the growth of a lung cancer cell line in the peritoneal cavity of mice.

The mesothelin (MSLN) gene encodes a 71 kilodalton (kDa) precursor protein that is processed into megakaryocytic potentiating factor (MPF), a 31 kDa protein that is secreted from the cell, and mature mesothelin (mMSLN), a 40 kDa cell surface protein. The mMSLN binds to CA125, an interaction that has been implicated in the intra-cavitary spread of mesothelioma and ovarian cancer. To better define the role of MPF and mMSLN, growth of the lung cancer cell line A549 was evaluated in immuno-deficient mice with inactivation of the Msln gene. We observed that Msln-/- mice xenografted with intraperitoneal A549 tumors survive significantly long than tumor-bearing Msln+/+ mice. When tumor-bearing Msln-/- mice are supplemented with recombinant MPF (and to a lesser extent mMSLN), most of this survival advantage is lost. These studies demonstrate that MPF and mMSLN have an important role in the growth of lung cancer cells in vivo and raise the possibility that inactivation of MPF may be a useful treatment for lung and other MSLN expressing cancers.

A recombinant immunotoxin engineered for increased stability by adding a disulfide bond has decreased immunogenicity.

Recombinant immunotoxins (RITs) are anti-cancer agents that combine the Fv of an antibody against cancer cells with a protein toxin from bacteria or plants. Since RITs contain a non-human protein, immunogenicity can be an obstacle in their development. In this study, we have explored the hypothesis that increasing stability can reduce the immunogenicity of a RIT using HA22-LR, which is composed of an anti-CD22 Fv fused to domain III of Pseudomonas exotoxin A. We introduced a disulfide bond into domain III by identifying and mutating two structurally adjacent residues to cysteines at sites suggested by computer modeling. This RIT, HA22-LR-DB, displays a remarkable increase in thermal stability and an enhanced resistance to trypsin degradation. In addition, HA22-LR-DB retains cytotoxic and anti-tumor activity, while exhibiting significantly lower immunogenicity in mice. This study demonstrates that it is possible to design mutations in a protein molecule that will increase the stability of the protein and thereby reduce its immunogenicity.

The influence of domain structures on the signal transduction of chimeric receptors derived from the erythropoietin receptor.

Although cytokine receptors regulate many cellular functions, contribution of receptor's domains and their conformation to signal transduction remains unclear. In this study, we designed a series of chimeric erythropoietin receptor (EpoR) variants encoding a haemagglutinin epitope-tagged anti-fluorescein single-chain Fv and different combinations of extracellular D1/D2 domain(s) of EpoR as the extracellular domain to allow the receptor to be activated by multiple ligands. Furthermore, one to four Ala residues were inserted at the intracellular juxtamembrane region of each chimeric receptor to modulate the conformation of the intracellular domain. When the chimeric receptors were expressed in Ba/F3 cells, cell-surface expression levels of chimeric receptors without D2 domain were markedly lowered, suggesting a role of D2 domain for stabilizing the receptor. Furthermore, the ligand-dependent cell proliferation was strongly affected by extracellular domain structures and the number of inserted Ala residues. Moreover, the conformational change of chimeric receptors was induced by various ligands to detect the phosphorylation of JAK2, STAT5 and ERK2, whose activations are characteristics of EpoR signalling. Consequently, the phosphorylation pattern of these signal transducers was significantly influenced by ligands and receptor variants. These results indicate that signal transduction of EpoR is strongly affected by conformation of both extracellular and intracellular domains.

Beam-width-dependent filtering properties of strong volume holographic gratings.

The finite dimension of the incident beam used to read out volume holographic gratings has interesting effects on their filtering properties. As the readout beam gets narrower, there is more deviation from the ideal response predicted for monochromatic plane waves. In this paper we experimentally explore beam-width-dependent phenomena such as wavelength selectivities, angular selectivities, and diffracted beam profiles. Volume gratings in both reflection and transmission geometries are investigated near 1550 nm. Numerical simulations utilizing the technique of Fourier decomposition provide a satisfactory explanation and confirm that the spread of spatial harmonics is the main contributing factor.

Diffraction from deformed volume holograms: perturbation theory approach.

We derive the response of a volume grating to arbitrary small deformations, using a perturbative approach. This result is of interest for two applications: (a) when a deformation is undesirable and one seeks to minimize the diffracted field's sensitivity to it and (b) when the deformation itself is the quantity of interest and the diffracted field is used as a probe into the deformed volume where the hologram was originally recorded. We show that our result is consistent with previous derivations motivated by the phenomenon of shrinkage in photopolymer holographic materials. We also present the analysis of the grating's response to deformation due to a point indenter and present experimental results consistent with theory.

Real-time spectral imaging in three spatial dimensions.

We report what is to our knowledge the first volume-holographic optical imaging instrument with the capability to return three-dimensional spatial as well as spectral information about semitranslucent microscopic objects in a single measurement. The four-dimensional volume-holographic microscope is characterized theoretically and experimentally by use of fluorescent microspheres as objects.

Volume holographic hyperspectral imaging.

A volume hologram has two degenerate Bragg-phase-matching dimensions and provides the capability of volume holographic imaging. We demonstrate two volume holographic imaging architectures and investigate their imaging resolution, aberration, and sensitivity. The first architecture uses the hologram directly as an objective imaging element where strong aberration is observed and confirmed by simulation. The second architecture uses an imaging lens and a transmission geometry hologram to achieve linear two-dimensional optical sectioning and imaging of a four-dimensional (spatial plus spectral dimensions) object hyperspace. Multiplexed holograms can achieve simultaneously three-dimensional imaging of an object without a scanning mechanism.

Folded shift multiplexing.

Shift multiplexing is a holographic recording method that uses a spherical reference wave. We extend the principle to a thin slab of holographic material that acts as a waveguide. Total internal reflection folds the reference spherical beam in one dimension. We demonstrate that the shift selectivity with the folded spherical beam is independent of the slab thickness but depends instead on the numerical aperture of the coupled spherical wave. A shift selectivity of 0.5 microm has been achieved with a 1-mm-thick LiNbO3 crystal and 50 high-definition data pages are recorded with this method.