Factor VIII trafficking to CD4+ T cells shapes its immunogenicity and requires several types of antigen-presenting cells.

Despite >80 years of clinical experience with coagulation factor VIII (FVIII) inhibitors, surprisingly little is known about the in vivo mechanism of this most serious complication of replacement therapy for hemophilia A. These neutralizing antidrug alloantibodies arise in ∼30% of patients. Inhibitor formation is T-cell dependent, but events leading up to helper T-cell activation have been elusive because of, in part, the complex anatomy and cellular makeup of the spleen. Here, we show that FVIII antigen presentation to CD4+ T cells critically depends on a select set of several anatomically distinct antigen-presenting cells, whereby marginal zone B cells and marginal zone and marginal metallophilic macrophages but not red pulp macrophages (RPMFs) participate in shuttling FVIII to the white pulp in which conventional dendritic cells (DCs) prime helper T cells, which then differentiate into follicular helper T (Tfh) cells. Toll-like receptor 9 stimulation accelerated Tfh cell responses and germinal center and inhibitor formation, whereas systemic administration of FVIII alone in hemophilia A mice increased frequencies of monocyte-derived and plasmacytoid DCs. Moreover, FVIII enhanced T-cell proliferation to another protein antigen (ovalbumin), and inflammatory signaling-deficient mice were less likely to develop inhibitors, indicating that FVIII may have intrinsic immunostimulatory properties. Ovalbumin, which, unlike FVIII, is absorbed into the RPMF compartment, fails to elicit T-cell proliferative and antibody responses when administered at the same dose as FVIII. Altogether, we propose that an antigen trafficking pattern that results in efficient in vivo delivery to DCs and inflammatory signaling, shape the immunogenicity of FVIII.

Intravital Microscopy Reveals Unforeseen Biodistribution Within the Liver and Kidney Mechanistically Connected to the Clearance of a Bifunctional Antibody.

Bifunctional antibody (BfAb) therapeutics offer the potential for novel functionalities beyond those of the individual monospecific entities. However, combining these entities into a single molecule can have unpredictable effects, including changes in pharmacokinetics that limit the compound's therapeutic profile. A better understanding of how molecular modifications affect in vivo tissue interactions could help inform BfAb design. The present studies were predicated on the observation that a BfAb designed to have minimal off-target interactions cleared from the circulation twice as fast as the monoclonal antibody (mAb) from which it was derived. The present study leverages the spatial and temporal resolution of intravital microscopy (IVM) to identify cellular interactions that may explain the different pharmacokinetics of the two compounds. Disposition studies of mice demonstrated that radiolabeled compounds distributed similarly over the first 24 hours, except that BfAb accumulated approximately two- to -three times more than mAb in the liver. IVM studies of mice demonstrated that both distributed to endosomes of liver endothelia but with different kinetics. Whereas mAb accumulated rapidly within the first hour of administration, BfAb accumulated only modestly during the first hour but continued to accumulate over 24 hours, ultimately reaching levels similar to those of the mAb. Although neither compound was freely filtered by the mouse or rat kidney, BfAb, but not mAb, was found to accumulate over 24 hours in endosomes of proximal tubule cells. These studies demonstrate how IVM can be used as a tool in drug design, revealing unpredicted cellular interactions that are undetectable by conventional analyses. SIGNIFICANCE STATEMENT: Bifunctional antibodies offer novel therapeutic functionalities beyond those of the individual monospecific entities. However, combining these entities into a single molecule can have unpredictable effects, including undesirable changes in pharmacokinetics. Studies of the dynamic distribution of a bifunctional antibody and its parent monoclonal antibody presented here demonstrate how intravital microscopy can expand our understanding of the in vivo disposition of therapeutics, detecting off-target interactions that could not be detected by conventional pharmacokinetics approaches or predicted by conventional physicochemical analyses.

Reducing target binding affinity improves the therapeutic index of anti-MET antibody-drug conjugate in tumor bearing animals.

Many oncology antibody-drug conjugates (ADCs) have failed to demonstrate efficacy in clinic because of dose-limiting toxicity caused by uptake into healthy tissues. We developed an approach that harnesses ADC affinity to broaden the therapeutic index (TI) using two anti-mesenchymal-epithelial transition factor (MET) monoclonal antibodies (mAbs) with high affinity (HAV) or low affinity (LAV) conjugated to monomethyl auristatin E (MMAE). The estimated TI for LAV-ADC was at least 3 times greater than the HAV-ADC. The LAV- and HAV-ADCs showed similar levels of anti-tumor activity in the xenograft model, while the 111In-DTPA studies showed similar amounts of the ADCs in HT29 tumors. Although the LAV-ADC has ~2-fold slower blood clearance than the HAV-ADC, higher liver toxicity was observed with HAV-ADC. While the SPECT/CT 111In- and 124I- DTPA findings showed HAV-ADC has higher accumulation and rapid clearance in normal tissues, intravital microscopy (IVM) studies confirmed HAV mAb accumulates within hepatic sinusoidal endothelial cells while the LAV mAb does not. These results demonstrated that lowering the MET binding affinity provides a larger TI for MET-ADC. Decreasing the affinity of the ADC reduces the target mediated drug disposition (TMDD) to MET expressed in normal tissues while maintaining uptake/delivery to the tumor. This approach can be applied to multiple ADCs to improve the clinical outcomes.

Single molecule fluorescence correlation spectroscopy of single apoptotic cells using a red-fluorescent caspase probe.

The detection of single molecules in single cells has enabled biochemical analyses to be conducted with high sensitivity and high temporal resolution. In this work, detection of apoptosis was studied by single molecule fluorescence correlation spectroscopy (FCS) in single living cells. Caspase activity was assayed using a new red fluorogenic probe that avoids the spectral overlap of green fluorescent probes and cell autofluorescence. This new probe, 2SBPO-Casp, was synthesized by coupling a water-soluble Nile Blue derivative (2SBPO) to an aspartic acid residue. Upon apoptosis induction and caspase activation, free 2SBPO dye is shown to accumulate inside the cell after probe cleavage. In previous work in our lab, single molecule fluorescence in single apoptotic cells was detected 45 min after induction using a rhodamine 110-based probe. However, significant statistical analysis was needed to exclude false positives. The use of 2SBPO-Casp overcomes the autofluorescence problem and offers a steady fluorescence signal. In our single molecule FCS measurements, Ramos cells were determined apoptotic on the basis of their correlation coefficient value (R(2)). Cells that contain an R(2) ≥ 0.65 were identified as highly correlated and therefore determined to be apoptotic. Single apoptotic cells identified in this manner were found as early as 30 min after induction and the number of apoptotic cells reached a peak value at the 3rd hour, which is consistent with other techniques. Using single molecule techniques and a new apoptosis probe, the temporal dynamics were elucidated with better sensitivity and resolution than in previous studies.

Epigenetic basis of diabetic vasculopathy.

Type 2 diabetes mellitus (T2DM) causes peripheral vascular disease because of which several blood-borne factors, including vital nutrients fail to reach the affected tissue. Tissue epigenome is sensitive to chronic hyperglycemia and is known to cause pathogenesis of micro- and macrovascular complications. These vascular complications of T2DM may perpetuate the onset of organ dysfunction. The burden of diabetes is primarily because of a wide range of complications of which nonhealing diabetic ulcers represent a major component. Thus, it is imperative that current research help recognize more effective methods for the diagnosis and management of early vascular injuries. This review addresses the significance of epigenetic processes such as DNA methylation and histone modifications in the evolution of macrovascular and microvascular complications of T2DM.

Early detection of apoptosis in living cells by fluorescence correlation spectroscopy.

Early detection of apoptotic cells via caspase activity is demonstrated with fast response time. Fluorescence correlation spectroscopy (FCS) is used to identify the presence of a cleaved fluorogenic probe based on the fluorescence of rhodamine 110 in Jurkat cells. FCS curves are shown to be markedly different for autofluorescent (non-apoptotic) cells, whereas cells with cleaved probe showed diffusion and molecular brightness characteristic of rhodamine 110. Using FCS measurements, cells were identified as apoptotic on the basis of the presence of autocorrelated fluorescence, average molecular brightness (eta), and molecular dwell time (tau (D)). Apoptotic cells identified in this manner were detected as early as 45 min after induction. Unlike other methods with similar identification times, such as western blotting and electron microscopy, cells remain viable for further analysis. This multi-parameter approach is rapid, flexible, and does not require transfection of the cells prior to analysis, enabling apoptosis to be identified early in a wide variety of cell types.

Temporal dynamics of receptor-induced apoptosis in an affinity microdevice.

The temporal dynamics of Fas-induced apoptosis is elucidated. Jurkat cells are captured on the affinity surface of a microdevice coated with anti-CD95, an antibody known to induce apoptosis in cells via the extrinsic (caspase 8) pathway. The timing of apoptosis induction is controlled by the binding of the cells to the surface. Once bound, the cells are continuously stained with the caspase probe, L-bisaspartic acid rhodamine 110 (D(2)R), and the fluorescence of the cells was monitored for 6 h by light microscopy. This approach normalizes the temporal dynamics for each cell, as the binding event is also the start of apoptosis. In addition to providing the number of apoptotic cells over time, the fluorescence of individual cells can be monitored, providing information about the timing of caspase activity in each cell. The rate of caspase cleavage of D(2)R in each cell is also measured and shows good agreement between the cells in a given population. The effects of the caspase inhibitor, z-VAD-FMK, on the timing of caspase activity are also investigated and are shown to dramatically slow the apoptotic process. In the future, other caspase probes could be used to provide additional information about the temporal dynamics of caspase activation. Additional techniques, such as fluorescence correlation spectroscopy, can be coupled to these methods to provide faster temporal response and help to elucidate the heterogeneity of the apoptosis process.

Mitochondrial depolarization and repolarization in the early stages of acetaminophen hepatotoxicity in mice.

Mitochondrial injury and depolarization are primary events in acetaminophen hepatotoxicity. Previous studies have shown that restoration of mitochondrial function in surviving hepatocytes, which is critical to recovery, is at least partially accomplished via biogenesis of new mitochondria. However, other studies indicate that mitochondria also have the potential to spontaneously repolarize. Although repolarization was previously observed only at a sub-hepatotoxic dose of acetaminophen, we postulated that mitochondrial repolarization in hepatocytes outside the centrilobular regions of necrosis might contribute to recovery of mitochondrial function following acetaminophen-induced injury. Our studies utilized longitudinal intravital microscopy of millimeter-scale regions of the mouse liver to characterize the spatio-temporal relationship between mitochondrial polarization and necrosis early in acetaminophen-induced liver injury. Treatment of male C57BL/6J mice with a single intraperitoneal 250 mg/kg dose of acetaminophen resulted in hepatotoxicity that was apparent histologically within 2 h of treatment, leading to 20 and 60-fold increases in serum aspartate aminotransferase and alanine aminotransferase, respectively, within 6 h. Intravital microscopy of the livers of mice injected with rhodamine123, TexasRed-dextran, propidium iodide and Hoechst 33342 detected centrilobular foci of necrosis within extended regions of mitochondrial depolarization within 2 h of acetaminophen treatment. Although regions of necrosis were more apparent 6 h after acetaminophen treatment, the vast majority of hepatocytes with depolarized mitochondria did not progress to necrosis, but rather recovered mitochondrial polarization within 6 h. Recovery of mitochondrial function following acetaminophen hepatotoxicity thus involves not only biogenesis of new mitochondria, but also repolarization of existing mitochondria. These studies also revealed a spatial distribution of necrosis and mitochondrial depolarization whose single-cell granularity is inconsistent with the hypothesis that communication between neighboring cells plays an important role in the propagation of necrosis during the early stages of APAP hepatotoxicity. Small islands of healthy, intact cells were frequently found surrounded by necrotic cells, and small islands of necrotic cells were frequently found surrounded by healthy, intact cells. Time-series studies demonstrated that these "islands", consisting in some cases of single cells, are persistent; over a period of hours, injury does not spread from individual necrotic cells to their neighbors.

Simultaneous cell capture and induction of apoptosis using an anti-CD95 affinity microdevice.

A microfluidic device is designed and demonstrated for the simultaneous capture and induction of apoptosis in Jurkat cells. In this unique case, the cell capture event initiates a biological process. The device features a single channel made from poly(dimethylsiloxane) sealed to a glass substrate. The channel is coated with a series of reagents used in affinity chromatography separations of cells. In this case, the antibody used to capture the cells is functional anti-CD95 which captures the cells by binding to the Fas receptor on the cell membrane and, at the same time, inducing apoptosis via the caspase 8 pathway. Cells retained on the surface of the channel are known to be induced to undergo apoptosis. Medium is flowed slowly through the channel to maintain cell viability while the cells undergo apoptosis. After 3 h, staining with Annexin V-PE and 7-AAD revealed that 43.5% of the cells bound to the anti-CD95 coated channel are apoptotic, whereas 7.9% of cultured Jurkat cells induced with anti-CD95 for 3 h and stained in the same way were determined to be apoptotic by flow cytometry. The device provides a method of determining when apoptosis is induced, maintaining cell viability for long-term analysis and observing cells in real time as they are exposed to reagents that affect apoptosis. In the future, the device will be an invaluable tool for the study of the temporal dynamics of apoptosis.

A Versatile, Portable Intravital Microscopy Platform for Studying Beta-cell Biology In Vivo.

The pancreatic islet is a complex micro-organ containing numerous cell types, including endocrine, immune, and endothelial cells. The communication of these systems is lost upon isolation of the islets, and therefore the pathogenesis of diabetes can only be fully understood by studying this organized, multicellular environment in vivo. We have developed several adaptable tools to create a versatile platform to interrogate ß-cell function in vivo. Specifically, we developed ß-cell-selective virally-encoded fluorescent protein biosensors that can be rapidly and easily introduced into any mouse. We then coupled the use of these biosensors with intravital microscopy, a powerful tool that can be used to collect cellular and subcellular data from living tissues. Together, these approaches allowed the observation of in vivo ß-cell-specific ROS dynamics using the Grx1-roGFP2 biosensor and calcium signaling using the GcAMP6s biosensor. Next, we utilized abdominal imaging windows (AIW) to extend our in vivo observations beyond single-point terminal measurements to collect longitudinal physiological and biosensor data through repeated imaging of the same mice over time. This platform represents a significant advancement in our ability to study ß-cell structure and signaling in vivo, and its portability for use in virtually any mouse model will enable meaningful studies of ß-cell physiology in the endogenous islet niche.

Fluorescence correlation spectroscopy: a review of biochemical and microfluidic applications.

Over the years fluorescence correlation spectroscopy (FCS) has proven to be a useful technique that has been utilized in several fields of study. Although FCS initially suffered from poor signal-to-noise ratios, the incorporation of confocal microscopy has overcome this drawback and transformed FCS into a sensitive technique with high figures of merit. In addition, tandem methods have evolved to include dual-color cross-correlation, total internal reflection fluorescence correlation, and fluorescence lifetime correlation spectroscopy combined with time-correlated single-photon counting. In this review, we discuss several applications of FSC for biochemical, microfluidic, and cellular investigations.

Pharmacological zinc and phytase supplementation enhance metallothionein mRNA abundance and protein concentration in newly weaned pigs.

The swine industry feeds pharmacological zinc (Zn) to newly weaned pigs to improve health. Because most swine diets are plant-based with a high phytic acid content, we hypothesized that adding phytase to diets could reduce the amount of Zn required to obtain beneficial responses. The role of metallothionein (MT) in Zn homeostasis could be important in this positive response. Thus, the goal of this study was to investigate the effect of dietary Zn and phytase on relative MT mRNA abundance and protein concentration in newly weaned pigs. Diets containing adequate (150 mg Zn/kg) or pharmacological concentrations of Zn (1000 or 2000 mg Zn/kg), as zinc oxide, with or without phytase [0, 500 phytase units (FTU)/kg, Natuphos, BASF] were fed in a 3 x 2 factorial design. Plasma and tissue minerals were measured in pigs killed after 14 d of dietary intervention. Hepatic and renal relative MT mRNA abundance and protein were greater (P < 0.05) in pigs fed 1000 mg Zn/kg with phytase, or 2000 mg Zn/kg with or without phytase vs. the remaining treatments. Intestinal mucosa MT mRNA abundance and protein were greater (P < 0.05) in pigs fed 2000 mg Zn/kg with phytase than in pigs fed 2000 mg Zn/kg alone or 1000 mg Zn/kg with phytase. Pigs fed 1000 mg Zn/kg plus phytase or 2000 mg Zn/kg with or without phytase had higher plasma, hepatic, and renal Zn than those fed the adequate Zn diets or 1000 mg Zn/kg. We conclude that feeding 1000 mg Zn/kg with phytase enhances MT mRNA abundance and protein and Zn absorption to the same degree as 2000 mg Zn/kg with and without phytase.