Highly Efficient SARS-CoV-2 Infection of Human Cardiomyocytes: Spike Protein-Mediated Cell Fusion and Its Inhibition.

Severe cardiovascular complications can occur in coronavirus disease of 2019 (COVID-19) patients. Cardiac damage is attributed mostly to the aberrant host response to acute respiratory infection. However, direct infection of cardiac tissue by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also occurs. We examined here the cardiac tropism of SARS-CoV-2 in spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These cardiomyocytes express the angiotensin-converting enzyme 2 (ACE2) receptor but not the transmembrane protease serine 2 (TMPRSS2) that mediates spike protein cleavage in the lungs. Nevertheless, SARS-CoV-2 infection of hiPSC-CMs was prolific; viral transcripts accounted for about 88% of total mRNA. In the cytoplasm of infected hiPSC-CMs, smooth-walled exocytic vesicles contained numerous 65- to 90-nm particles with canonical ribonucleocapsid structures, and virus-like particles with knob-like spikes covered the cell surface. To better understand how SARS-CoV-2 spreads in hiPSC-CMs, we engineered an expression vector coding for the spike protein with a monomeric emerald-green fluorescent protein fused to its cytoplasmic tail (S-mEm). Proteolytic processing of S-mEm and the parental spike were equivalent. Live cell imaging tracked spread of S-mEm cell-to-cell and documented formation of syncytia. A cell-permeable, peptide-based molecule that blocks the catalytic site of furin and furin-like proteases abolished cell fusion. A spike mutant with the single amino acid change R682S that disrupts the multibasic furin cleavage motif was fusion inactive. Thus, SARS-CoV-2 replicates efficiently in hiPSC-CMs and furin, and/or furin-like-protease activation of its spike protein is required for fusion-based cytopathology. This hiPSC-CM platform enables target-based drug discovery in cardiac COVID-19. IMPORTANCE Cardiac complications frequently observed in COVID-19 patients are tentatively attributed to systemic inflammation and thrombosis, but viral replication has occasionally been confirmed in cardiac tissue autopsy materials. We developed an in vitro model of SARS-CoV-2 spread in myocardium using induced pluripotent stem cell-derived cardiomyocytes. In these highly differentiated cells, viral transcription levels exceeded those previously documented in permissive transformed cell lines. To better understand the mechanisms of SARS-CoV-2 spread, we expressed a fluorescent version of its spike protein that allowed us to characterize a fusion-based cytopathic effect. A mutant of the spike protein with a single amino acid mutation in the furin/furin-like protease cleavage site lost cytopathic function. Of note, the fusion activities of the spike protein of other coronaviruses correlated with the level of cardiovascular complications observed in infections with the respective viruses. These data indicate that SARS-CoV-2 may cause cardiac damage by fusing cardiomyocytes.

Differential effect of amyloid beta peptides on mitochondrial axonal trafficking depends on their state of aggregation and binding to the plasma membrane.

Inhibition of mitochondrial axonal trafficking by amyloid beta (Aß) peptides has been implicated in early pathophysiology of Alzheimer's Disease (AD). Yet, it remains unclear whether the loss of motility inevitably induces the loss of mitochondrial function, and whether restoration of axonal trafficking represents a valid therapeutic target. Moreover, while some investigations identify Aß oligomers as the culprit of trafficking inhibition, others propose that fibrils play the detrimental role. We have examined the effect of a panel of Aß peptides with different mutations found in familial AD on mitochondrial motility in primary cortical mouse neurons. Peptides with higher propensity to aggregate inhibit mitochondrial trafficking to a greater extent with fibrils inducing the strongest inhibition. Binding of Aß peptides to the plasma membrane was sufficient to induce trafficking inhibition where peptides with reduced plasma membrane binding and internalization had lesser effect on mitochondrial motility. We also found that Aß peptide with Icelandic mutation A673T affects axonal trafficking of mitochondria but has very low rates of plasma membrane binding and internalization in neurons, which could explain its relatively low toxicity. Inhibition of mitochondrial dynamics caused by Aß peptides or fibrils did not instantly affect mitochondrial bioenergetic and function. Our results support a mechanism where inhibition of axonal trafficking is initiated at the plasma membrane by soluble low molecular weight Aß species and is exacerbated by fibrils. Since trafficking inhibition does not coincide with the loss of mitochondrial function, restoration of axonal transport could be beneficial at early stages of AD progression. However, strategies designed to block Aß aggregation or fibril formation alone without ensuring the efficient clearance of soluble Aß may not be sufficient to alleviate the trafficking phenotype.

Cell Damage in Light Chain Amyloidosis: FIBRIL INTERNALIZATION, TOXICITY AND CELL-MEDIATED SEEDING.

Light chain (AL) amyloidosis is an incurable human disease characterized by the misfolding, aggregation, and systemic deposition of amyloid composed of immunoglobulin light chains (LC). This work describes our studies on potential mechanisms of AL cytotoxicity. We have studied the internalization of AL soluble proteins and amyloid fibrils into human AC16 cardiomyocytes by using real time live cell image analysis. Our results show how external amyloid aggregates rapidly surround the cells and act as a recruitment point for soluble protein, triggering the amyloid fibril elongation. Soluble protein and external aggregates are internalized into AC16 cells via macropinocytosis. AL amyloid fibrils are shown to be highly cytotoxic at low concentrations. Additionally, caspase assays revealed soluble protein induces apoptosis, demonstrating different cytotoxic mechanisms between soluble protein and amyloid aggregates. This study emphasizes the complex immunoglobulin light chain-cell interactions that result in fibril internalization, protein recruitment, and cytotoxicity that may occur in AL amyloidosis.

Unique molecular signatures of Alzheimer's disease amyloid ß peptide mutations and deletion during aggregate/oligomer/fibril formation.

The formation of amyloid ß (Aß) peptide aggregates, oligomers, and fibrils is a dynamic process; however, the kinetics of their formation is not well understood. This study compares the time course of aggregate/fibril formation by transmission electron microscopy (TEM) analyses with that of oligomer/fibril formation by Western blot analysis under native and denaturing conditions. Efforts to deaggregate/defibrillate these peptides by using hexafluoroisopropanol, ammonium hydroxide, or dimethylsulfoxide did not change the nondenaturing polyacrylamide gel electrophoresis (PAGE) footprints or drive the peptides to a monomeric species. Regardless of the pretreatment protocol, TEM analyses reveal that all Aß peptides (Aß40, Aß42, Aß39E22Δ [Osaka], Aß40E22G [Arctic], Aß40E22Q [Dutch], and Aß40A2T [Icelandic]) immediately formed nonfibrillar, amorphous aggregates when first placed into solution with the Osaka mutation, quickly forming early-stage fibrils. The extent of fibril formation for other Aß peptides is time dependent, with the Arctic mutation forming fibrils at 1 hr, the Dutch and Icelandic at 4 hr, Aß42 at 8 hr, and Aß40 at 24 hr. In contrast, nondenaturing PAGE revealed unique footprints for the different Aß species. The rapidity of aggregate formation and the rapid transition to fibrils, particularly for the Osaka deletion, suggest an important role for aggregates/fibrils of Aß in the development of neuronal degeneration.

Role of mutations in the cellular internalization of amyloidogenic light chains into cardiomyocytes.

Light chain (AL) amyloidosis is characterized by the misfolding of immunoglobulin light chains, accumulating as amyloid fibrils in vital organs. Multiple reports have indicated that amyloidogenic light chains internalize into a variety of cell types, but these studies used urine-derived proteins without indicating any protein sequence information. As a result, the role of somatic mutations in amyloidogenic protein internalization has not been yet studied. We characterized the internalization of AL-09, an AL amyloidosis protein into mouse cardiomyocytes. We also characterized the internalization of the germline protein κI O18/O8, devoid of somatic mutations, and three AL-09 restorative mutations (I34N, Q42K, and H87Y) previously characterized for their role in protein structure, stability, and amyloid formation kinetics. All proteins shared a common internalization pathway into lysosomal compartments. The proteins caused different degrees of lysosomal expansion. Oregon green (OG) labeled AL-09 showed the most rapid internalization, while OG-Q42K presented the slowest rate of internalization.

Alzheimer's disease amyloid ß-protein mutations and deletions that define neuronal binding/internalization as early stage nonfibrillar/fibrillar aggregates and late stage fibrils.

Accumulation of amyloid ß-protein (Aß) in neurons has been demonstrated to precede its formation as amyloid plaques in the extracellular space in Alzheimer's disease (AD) patients. Consequently, intraneuronal Aß accumulation is thought to be a critical first step in the fatal cascade of events that leads to neuronal degeneration in AD. Understanding the structural basis of neuronal binding and uptake of Aß might lead to potential therapeutic targets that could block this binding and the subsequent neurodegeneration that leads to the pathogenesis of AD. Previously, we demonstrated that mutation of the two adjacent histidine residues of Aß40 (H13,14G) resulted in a significant decrease in its level of binding to PC12 cells and mouse cortical/hippocampal neurons. We now demonstrate that the weakened neuronal binding follows the mutation order of H13G < H14G < H13,14G, which suggests that the primary domain for neuronal binding of Aß40 involves histidine at position 13. A novel APP mutation (E693Δ) that produced a variant Aß lacking glutamate 22 (E22Δ) in Japanese pedigrees was recently identified to have AD-type dementia without amyloid plaque formation but with extensive intraneuronal Aß in transfected cells and transgenic mice expressing this deletion. Deletion of glutamate 22 of Aß40 resulted in a 6-fold enhancement of PC12 neuronal binding that was not decreased by the H13G mutation. The dose-dependent enhanced binding of E22Δ explains the high level of intraneuronal Aß seen in this pedigree. Fluorescence anisotropy experiments at room temperature showed very rapid aggregation with increased tyrosine rigidity of Aß39E22Δ, Aß41E22Δ, and Aß42 but not Aß40. This rigidity was decreased but not eliminated by prior treatment with dimethyl sulfoxide. Surprisingly, all peptides showed an aggregated state when evaluated by transmission electron microscopy, with Aß39E22Δ having early stage fibrils, which was also verified by atomic force microscopy. This aggregation was not affected by centrifugation or pretreatment with organic solvents. The enhanced neuronal binding of Aß, therefore, results from aggregate binding to neurons, which requires H13 for Aß40 but not for E22Δ or Aß42. These latter proteins display increased tyrosine rigidity that likely masks the H13 residue, or alternatively, the H13 residue is not required for neuronal binding of these proteins as it is for Aß40. Late state fibrils also showed enhanced neuronal binding for E22Δ but not Aß40 with subsequent intraneuronal accumulation in lysosomes. This suggests that there are multiple pathways of binding/internalization for the different Aß proteins and their aggregation states or fibrillar structure.

Myocilin levels in primary open-angle glaucoma and pseudoexfoliation glaucoma human aqueous humor.

PURPOSE: To determine the concentration of myocilin in primary open-angle glaucoma (POAG) and pseudoexfoliation glaucoma (PEXG) aqueous humor. METHODS: Aqueous humor was collected during surgery from patients with POAG, PEXG, and elective cataract removal (control). Volume-equivalent aqueous samples were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gradient gels. Quantification of myocilin levels was performed using Western blots probed with 2 independent N-terminal polyclonal anti-myocilin antibodies (AB1 and AB2) followed by densitometry. Myocilin levels in aqueous humor were quantified by plotting the densitometry readings of the aqueous samples against a recombinant myocilin standard curve. Total protein concentration was determined by Bradford protein assay. Transforming growth factor ß 2 levels were assessed by enzyme-linked immunosorbent assay. RESULTS: Myocilin levels are significantly elevated in human POAG aqueous humor when compared with control aqueous humor (AB1: 0.66±0.53 ng/µL vs. 0.23±0.20 ng/µL, P<0.001; AB2: 0.98±0.59 ng/µL vs. 0.65±0.5 ng/µL, P<0.03; mean±SD). Myocilin makes up a larger percent of the total protein in POAG aqueous humor compared with control aqueous (AB1: 0.26±0.20% vs. 0.10±0.20%, P<0.001; AB2: 0.43±0.32% vs. 0.28±0.18%, P<0.05). In contrast to POAG, myocilin levels were not elevated in PEXG aqueous humor when compared with control aqueous humor. No correlation between myocilin and transforming growth factor ß 2 levels was observed. CONCLUSIONS: Myocilin is elevated in POAG, but not in PEXG aqueous humor.

HH domain of Alzheimer's disease Abeta provides structural basis for neuronal binding in PC12 and mouse cortical/hippocampal neurons.

A key question in understanding AD is whether extracellular Abeta deposition of parenchymal amyloid plaques or intraneuronal Abeta accumulation initiates the AD process. Amyloid precursor protein (APP) is endocytosed from the cell surface into endosomes where it is cleaved to produce soluble Abeta which is then released into the brain interstitial fluid. Intraneuronal Abeta accumulation is hypothesized to predominate from the neuronal uptake of this soluble extracellular Abeta rather than from ER/Golgi processing of APP. We demonstrate that substitution of the two adjacent histidine residues of Abeta40 results in a significant decrease in its binding with PC12 cells and mouse cortical/hippocampal neurons. These substitutions also result in a dramatic enhancement of both thioflavin-T positive fibril formation and binding to preformed Abeta fibrils while maintaining its plaque-binding ability in AD transgenic mice. Hence, alteration of the histidine domain of Abeta prevented neuronal binding and drove Abeta to enhanced fibril formation and subsequent amyloid plaque deposition--a potential mechanism for removing toxic species of Abeta. Substitution or even masking of these Abeta histidine residues might provide a new therapeutic direction for minimizing neuronal uptake and subsequent neuronal degeneration and maximizing targeting to amyloid plaques.

Surface plasmon resonance binding kinetics of Alzheimer's disease amyloid beta peptide-capturing and plaque-binding monoclonal antibodies.

Several different monoclonal antibodies (mAbs) have been actively developed in the field of Alzheimer's disease (AD) for basic science and clinical applications; however, the binding kinetics of many of the mAbs with the beta-amyloid peptides (Abeta) are poorly understood. A panel of mAbs with different Abeta recognition sites, including our plaque-binding antibody (IgG4.1), a peptide-capturing antibody (11A50), and two classical mAbs (6E10 and 4G8) used for immunohistochemistry, were chosen for characterization of their kinetics of binding to monomeric and fibrillar forms of Abeta40 using surface plasmon resonance and their amyloid plaque binding ability in AD mouse brain sections using immunohistochemistry. The plaque-binding antibody (IgG4.1) with epitope specificity of Abeta(2-10) showed a weaker affinity (512 nM) for monomeric Abeta40 but a higher affinity (1.5 nM) for Abeta40 fibrils and labeled dense core plaques better than 6E10 as determined by immunohistochemistry. The peptide-capturing antibody (11A50) showed preferential affinity (32.5 nM) for monomeric Abeta40 but did not bind to Abeta40 fibrils, whereas antibodies 6E10 and 4G8 had moderate affinity for monomeric Abeta40 (22.3 and 30.1 nM, respectively). 4G8, which labels diffuse plaques better than 6E10, had a higher association rate constant than 6E10 but showed similar association and dissociation kinetics compared to those of 11A50. Enzymatic digestion of IgG4.1 to the F(ab')(2)4.1 fragments or their polyamine-modified derivatives that enhance blood-brain barrier permeability did not affect the kinetic properties of the antigen binding site. These differences in kinetic binding to monomeric and fibrillar Abeta among various antibodies could be utilized to distinguish mAbs that might be useful for immunotherapy or amyloid plaque imaging versus those that could be utilized for bioanalytical techniques.

Characterization of monoclonal antibodies against the glaucoma-associated protein myocilin.

Although the glaucoma-associated protein myocilin has been the focus of intensive research, its biological function is still unknown. One of the limiting factors has been the lack of well-characterized antibodies, particularly monoclonal antibodies. We describe the development of six monoclonal antibodies specific to myocilin and characterize their suitability in Western blot and immunohistochemical applications. Three of the six monoclonal antibodies recognize the N-terminus of myocilin (amino acids 33-214), two antibodies recognize the middle third of the protein (amino acids 215-368), and one antibody recognizes the C-terminus (amino acids 369-504). Isotyping revealed that all antibodies are of the IgG1 kappa class except one, which is IgG2b kappa. Purified myocilin monoclonal antibodies were able to recognize myocilin in human aqueous humor separated on denatured/reduced and native gels, and human trabecular meshwork lysate by Western blot. Myocilin was also detected by immunohistochemistry in trabecular meshwork, ciliary body, iris, cornea, sclera, choroid, and retinal pigment epithelial cells.

Prostaglandins increase trabecular meshwork outflow facility in cultured human anterior segments.

PURPOSE: To determine the effect of latanoprost free acid and prostaglandin E1 (PGE1) on outflow facility in cultured human anterior segments. Clinical studies find prostaglandin treatment increases uveoscleral outflow, but do not agree whether trabecular outflow increases. Cultured anterior segments eliminate the uveoscleral pathway and enable a direct assessment of trabecular outflow. DESIGN: Laboratory investigation. METHODS: One anterior segment of an eye pair was placed in perfusion organ culture and received a continuous infusion of drug while the fellow anterior segment received vehicle. Histologic changes were assessed. Zymography and Western blots were used to analyze matrix metalloprotease (MMP) activity. Scleral hydraulic conductivities were measured. RESULTS: Latanoprost significantly increased outflow facility (67% +/- 11% vs control 6% +/- 10%, P < .001). Facility changes occurred within one hour of receiving drug, reaching a new baseline by 24 hours. Facility changes were reversible, requiring about 48 hours to return to pre-drug values. PGE1 caused less facility change (13% +/- 17% vs control 1% +/- 11%, P = .02). Prostaglandin treated anterior segments had regions of focal detachment and loss of Schlemm canal endothelial cells, with loss of extracellular matrix underlying some areas. MMPs were not consistently increased in Western blots, zymography, or immunohistochemistry. Scleral hydraulic conductivity increased, but not enough to account for total facility increase. CONCLUSIONS: Prostaglandins increase outflow facility in perfusion organ culture of human anterior segments. MMP activity was not consistently increased, and scleral hydraulic conductivity was not increased sufficiently to account for total facility increase. The histologic changes suggest a direct trabecular meshwork effect.

Perfusion of his-tagged eukaryotic myocilin increases outflow resistance in human anterior segments in the presence of aqueous humor.

PURPOSE: A previous study by the authors has shown that recombinant myocilin purified from a prokaryotic expression system increases outflow resistance in cultured human anterior segments. The present study was performed to determine whether full-length myocilin purified from a human trabecular meshwork cell expression system alters outflow resistance after infusion into human anterior segments. METHODS: A feline immunodeficiency virus vector encoding both full-length myocilin (amino acids 1-503 fused to C-terminal V5 and six-histidine epitopes) and puromycin resistance was used to transduce a transformed trabecular meshwork cell line (TM5). Stably expressing cells were selected with puromycin. Recombinant myocilin was purified from the media using nickel ion affinity chromatography. Control purifications were performed on media from parental TM5 cells. Anterior segments of human eyes were placed in organ culture and perfused with either Dulbecco's modified Eagle's medium (DMEM) or DMEM supplemented with 50% porcine aqueous humor. One eye received an anterior chamber exchange with recombinant myocilin (2 microg/mL), whereas the fellow eye received an equal volume of control. Immunohistochemistry was performed with anti-myocilin and anti-V5 antibodies. Native polyacrylamide gel electrophoresis was used to analyze myocilin complex formation in porcine aqueous humor. RESULTS: Recombinant myocilin in porcine aqueous humor increased outflow resistance in cultured human anterior segments (91% +/- 68% [mean +/- SD] versus 18% +/- 31% in fellow control eye; n = 9, P = 0.004). Maximum outflow resistance was obtained 5 to 17 hours after infusion and remained above baseline for >3 days. Recombinant myocilin also increased outflow resistance in eyes incubated in DMEM, but only if myocilin was preincubated with porcine aqueous humor (78% +/- 77% when preincubated in DMEM containing porcine aqueous humor versus 13% +/- 15% when preincubated with DMEM alone, n = 6, P = 0.03). Recombinant myocilin appears to form a complex in porcine aqueous humor with a heat-labile protein(s). Immunohistochemistry revealed the presence of myocilin in the juxtacanalicular region of the trabecular meshwork. CONCLUSIONS: Myocilin purified from human trabecular meshwork cells increased outflow resistance in cultured human anterior segments, but only after incubation with porcine aqueous humor. Recombinant myocilin appears to form a complex in porcine aqueous humor that enables it to bind specifically within the trabecular meshwork.

Primary trabecular meshwork cells incubated in human aqueous humor differ from cells incubated in serum supplements.

PURPOSE: To determine whether aqueous humor, the in vivo source of nutrients for trabecular meshwork cells, alters cellular and molecular characteristics in primary trabecular monolayer cell cultures when compared with standard culture conditions. METHODS: Human primary trabecular meshwork cell cultures were grown in DMEM supplemented with 50% human aqueous humor (DMEM-AH), heat-denatured DMEM-AH, 10% fetal bovine serum (DMEM-FBS, the standard culture supplement), or heat-denatured DMEM-FBS. Confluent trabecular cells were assayed for cell propagation and morphology for 21 days. Protein expression profiles of trabecular cell lysates were analyzed by two-dimensional polyacrylamide gel electrophoresis. Western blot analysis was used to determine the protein expression of myocilin and TIMP-1 in conditioned media collected from trabecular cells at 5, 10, 15, and 21 days. Myocilin expression was also analyzed by Western immunoblots after addition of dexamethasone (10(-7) M) or ascorbic acid (29 mg/dL). RESULTS: Trabecular cells supplemented with DMEM-AH for 21 days showed decreased cell proliferation when compared with DMEM-FBS (11% vs. 141%). Cellular morphology was also altered: Trabecular cells incubated in DMEM-AH showed larger-, broader-, and flatter-appearing cells than did the more spindle-shaped cells grown in DMEM-FBS. Protein profiles of trabecular cell lysates isolated from cells incubated in DMEM-AH differed from those incubated in DMEM-FBS. In DMEM-AH-conditioned medium, myocilin expression was increased and TIMP-1 expression was decreased at day 21. Induction of myocilin by dexamethasone was observed in conditioned medium isolated from cells treated with DMEM-FBS (442%), but only a 10% increase in myocilin was observed beyond the normal induction in DMEM-AH. Daily administration of ascorbic acid to DMEM-AH failed to increase myocilin expression beyond that obtained with DMEM-AH. CONCLUSIONS: Addition of human aqueous humor rather than FBS to trabecular monolayer cell cultures triggers significant changes in cellular and molecular characteristics. The protein component of aqueous humor is responsible for these changes. Aqueous humor supplementation may maintain cultured trabecular cells in a more physiologic state.

Unintegrated lentivirus DNA persistence and accessibility to expression in nondividing cells: analysis with class I integrase mutants.

The circumstances under which unintegrated lentivirus DNA can persist and be a functional template for transcription and protein expression are not clear. We constructed and validated the first class I (nonpleiotropic) integrase (IN) mutants for a non-human lentivirus (feline immunodeficiency virus [FIV]) and analyzed both these and known class I human immunodeficiency virus type 1 IN mutants. The FIV IN mutants (D66V and D66V/D118A) had class I properties: Gag/Pol precursor expression, proteolytic processing, particle formation, and reverse transcriptase (RT) production were normal, while the transduction of dividing fibroblasts was prevented and integration was blocked. When injected into rat retinas, the wild-type (WT) vector produced extensive, persistent transgene expression, compared with only rare positive neuronal cells for the IN mutant vector. In contrast, both WT and mutant vectors produced entirely equivalent, effective transduction levels of primary rat neurons (retinal ganglion cells). By testing the hypothesis that the unexpected retinal neuron transduction was related to cell cycle status, we found that when fibroblasts were growth arrested, transduction and internally promoted transgene expression were not inhibited at all by the class I FIV or HIV-1 IN mutations. Cells were then transduced under aphidicolin arrest and were released from the block 48 h later. Vector expression was stable and durable during repeated passaging in WT vector-transduced cells, while the release of cells transduced with equivalent RT units of class I IN mutant FIV or HIV vector resulted in a steady decline of expression, from 97 to 0% of cells by day 10. Southern blot and PCR analyses showed a lack of integration, irrespective of cell cycle, for the class I mutants and an increase in one- and two-long terminal repeat circular and linear unintegrated DNAs in growth-arrested cells. We conclude that if cell division is prevented, unintegrated FIV and HIV-1 vector DNAs can produce high-level internally promoted transgene expression equivalent to WT vectors. The expression correlates with the unintegrated DNA levels. These observations may facilitate the study of the roles of IN and other preintegration complex components in preintegration phases of infection by (i) providing an alternative way to monitor unintegrated nuclear cDNA forms, (ii) restricting ascertainment to the transcriptionally functional subset of unintegrated DNA, (iii) enabling analysis in individual, nondividing cells, and (iv) uncoupling other potential functions of IN from integration.

Phase 1 study of interleukin-12 in combination with rituximab in patients with B-cell non-Hodgkin lymphoma.

Rituximab is a chimeric murine/human monoclonal antibody that binds to CD20 on B lymphocytes. Although binding of the Fab domain may induce apoptosis, the Fc domain recruits immune effector functions to mediate cell lysis. Interleukin-12 (IL-12) facilitates cytolytic T-cell responses, enhances the lytic activity of natural killer (NK) cells, and induces the secretion of interferon gamma (IFN-gamma) by both T and NK cells. Therefore, the hypothesis was considered that combining IL-12 with rituximab would augment the immune-mediated cell lysis induced by rituximab. A phase 1 study of IL-12 in combination with rituximab was conducted in 43 adults with B-cell lymphoma to determine the optimal immunologic dose of this combination. Rituximab was administered at a dose of 375 mg/m(2) by intravenous infusion weekly for 4 weeks, and IL-12 was given subcutaneously twice weekly. The starting dose of IL-12 was 30 ng/kg and this was escalated to 500 ng/kg. Constitutional symptoms and liver enzyme elevations at 500 ng/kg of IL-12 were dose limiting. A greater than 20-fold increase in the serum levels of IFN-gamma and a 2.5- to 5-fold increase in inducible protein 10 (IP-10) levels was seen at IL-12 doses of 100 ng/kg or greater. Objective responses occurred in 29 of the 43 patients (69%), with 8 of 11 complete responses seen at IL-12 doses of 300 ng/kg or greater. The optimal immunologic dose of IL-12 in combination with rituximab was determined to be 300 ng/kg subcutaneously twice weekly starting on day 2. These data suggest that IL-12 and rituximab is an active combination and further studies of this combination in B-cell non-Hodgkin lymphoma are warranted.