Human iPSC-derived photoreceptor transplantation in the cone dominant 13-lined ground squirrel.

Several retinal degenerations affect the human central retina, which is primarily comprised of cones and is essential for high acuity and color vision. Transplanting cone photoreceptors is a promising strategy to replace degenerated cones in this region. Although this approach has been investigated in a handful of animal models, commonly used rodent models lack a cone-rich region and larger models can be expensive and inaccessible, impeding the translation of therapies. Here, we transplanted dissociated GFP-expressing photoreceptors from retinal organoids differentiated from human induced pluripotent stem cells into the subretinal space of damaged and undamaged cone-dominant 13-lined ground squirrel eyes. Transplanted cell survival was documented via noninvasive high-resolution imaging and immunohistochemistry to confirm the presence of human donor photoreceptors for up to 4 months posttransplantation. These results demonstrate the utility of a cone-dominant rodent model for advancing the clinical translation of cell replacement therapies.

Hepatic artery flow, inspired oxygen, and hemoglobin determine liver tissue saturation measured with visible diffuse reflectance spectroscopy (vis-DRS) in an in vivo swine model.

BACKGROUND: Prompt diagnosis of vascular compromise following pediatric liver transplantation and restoration of oxygen delivery to the liver improves organ survival. vis-DRS allows for real-time measurement of liver tissue saturation. METHODS: The current study used vis-DRS to determine changes in liver saturation during clinically relevant conditions of reduced oxygen delivery. In an in vivo swine model (n = 15), we determined liver tissue saturation (St O2 ) during stepwise reduction in hepatic artery flow, different inspiratory oxygen fraction (FiO2 ), and increasing hemodilution. A custom vis-DRS probe was placed directly on the organ. RESULTS: Liver tissue saturation decreased significantly with a decrease in hepatic artery flow. A reduction in hepatic artery flow to 25% of baseline reduced the St O2 by 15.3 ± 1.4% at FiO2 0.3 (mean ± SE, p < .0013), and by 8.3 ± 1.9% at FiO2 1.0 (p = .0013). After hemodilution to 7-8 g/dl, St O2 was reduced by 31.8% ± 2.7%, p < .001 (FiO2 0.3) and 26.6 ± 2.7%, p < .001 (FiO2 : 1.0) respectively. Portal venous saturation during low hepatic artery flow was consistently higher at FiO2 1.0. The gradient between portal venous saturation and liver tissue saturation was consistently greater at lower hemoglobin levels (7.0 ± 1.6% per g/dl hemoglobin, p < .001). CONCLUSIONS: Vis-DRS showed prompt changes in liver tissue saturation with decreases in hepatic artery blood flow. At hepatic artery flows below 50% of baseline, liver saturation depended on FiO2 and hemoglobin concentration suggesting that during hepatic artery occlusion, packed red blood cell transfusion and increased FiO2 may be useful measures to reduce hypoxic damage until surgical revascularization.

Optical Coherence Tomography Angiography in the Thirteen-Lined Ground Squirrel.

Purpose: To assess the performance of two spectral-domain optical coherence tomography-angiography systems in a natural model of hypoperfusion: the hibernating thirteen-lined ground squirrel (13-LGS). Methods: Using a high-speed (130 kHz) OCT-A system (HS-OCT-A) and a commercial OCT (36 kHz; Bioptigen Envisu; BE-OCT-A), we imaged the 13-LGS retina throughout its hibernation cycle. Custom software was used to extract the superior, middle, and deep capillary plexus (SCP, MCP, and DCP, respectively). The retinal vasculature was also imaged with adaptive optics scanning light ophthalmoscopy (AOSLO) during torpor to visualize individual blood cells. Finally, correlative histology with immunolabeled or DiI-stained vasculature was performed. Results: During euthermia, vessel density was similar between devices for the SCP and MCP (P = 0.88, 0.72, respectively), with a small difference in the DCP (-1.63 ± 1.54%, P = 0.036). Apparent capillary dropout was observed during torpor, but recovered after forced arousal, and this effect was exaggerated in high-speed OCT-A imaging. Based on cell flux measurements with AOSLO, increasing OCT-A scan duration by ∼1000× would avoid the apparent capillary dropout artifact. High correspondence between OCT-A (during euthermia) and histology enabled lateral scale calibration. Conclusions: While the HS-OCT-A system provides a more efficient workflow, the shorter interscan interval may render it more susceptible to the apparent capillary dropout artifact. Disambiguation between capillary dropout and transient ischemia can have important implications in the management of retinal disease and warrants additional diagnostics. Translational Relevance: The 13-LGS provides a natural model of hypoperfusion that may prove valuable in modeling the utility of OCT-A in human pathologies associated with altered blood flow.

Evaluation of visible diffuse reflectance spectroscopy in liver tissue: validation of tissue saturations using extracorporeal circulation.

SIGNIFICANCE: Real-time information about oxygen delivery to the hepatic graft is important to direct care and diagnose vascular compromise in the immediate post-transplant period. AIM: The current study was designed to determine the utility of visible diffuse reflectance spectroscopy (vis-DRS) for measuring liver tissue saturation in vivo. APPROACH: A custom-built vis-DRS probe was calibrated using phantoms with hemoglobin (Hb) and polystyrene microspheres. Ex vivo (extracorporeal circulation) and in vivo protocols were used in a swine model (n = 15) with validation via blood gas analysis. RESULTS: In vivo absorption and scattering measured by vis-DRS with and without biliverdin correction correlated closely between analyses. Lin's concordance correlation coefficients are 0.991 for µa and 0.959 for µs ' . Hb measured by blood test and vis-DRS with (R2 = 0.81) and without (R2 = 0.85) biliverdin correction were compared. Vis-DRS data obtained from the ex vivo protocol plotted against the PO2 derived from blood gas analysis showed a good fit for a Hill coefficient of 1.67 and P50 = 34 mmHg (R2 = 0.81). A conversion formula was developed to account for the systematic deviation, which resulted in a goodness-of-fit R2 = 0.76 with the expected oxygen dissociation curve. CONCLUSIONS: We show that vis-DRS allows for real-time measurement of liver tissue saturation, an indicator for liver perfusion and oxygen delivery.

Assessing Accumulation of Organic Material on Rodent Cage Accessories.

According to the 8th edition of the Guide for the Care and Use of Laboratory Animals (the Guide), rodent cage accessories, such as filter tops, should be sanitized at least once every 2 wk. We performed a study to test the hypothesis that organic contamination (measured by ATP content, expressed as relative light units (RLU)) of cage accessories (wire bar inserts and filter top lids) does not differ at 2 wk (14 d) as compared with 30, 60, and 90-d time points after cage change even when in constant use. An additional time point for filter top lids of 180 d after cage change was also evaluated. Eight groups were studied: the wire bar inserts and filter top lids used for mice and rats, in both static and individually ventilated cages (IVC). When analyzing data from both mouse and rat static and IVC caging, we found that the mean RLU values for mouse IVC and rat static and IVC cage components were below 100,000 RLU at the 14-d time point. The mean value for the mouse static group was slightly above 100,000 RLU at this time point. Based on this observation, we considered 100,000 RLU to be an appropriate actionable level. We concluded that changing wire bar inserts at least every 14 d, as recommended in the Guide for sanitizing these components in mouse and rat static cages, may be considered acceptable. This interval could be extended for mouse and rat IVC cages up to 90 d while remaining below this limit. Filter top lids for mouse static cages should be changed at least every 30 d, but static rat and IVC mouse/rat filter top lids could be changed up to every 180 d, while still staying below this actionable level of contamination.

Effect of Nearby Construction Activity on Endothelial Function, Sensitivity to Nitric Oxide, and Potassium Channel Activity in the Middle Cerebral Arteries of Rats.

The present study assessed the effect of nearby construction activity on the responses of rat middle cerebral arteries (MCA)to the endothelium-dependent vasodilator acetylcholine and the NO donor sodium nitroprusside (SNP) and the activity of MaxiK potassium channels in MCA smooth muscle cells from male Sprague-Dawley rats. Two monitoring systems were used to assess vibrations in the animal rooms during and immediately after construction activities near the research building where the animal facility is located. One was a commercially available system; the other was a Raspberry-Pi (RPi)-based vibration monitoring system designed in our laboratory that included a small computing unit attached to a rolling sensor (low sensitivity) and a piezoelectric film sensor (high sensitivity). Both systems recorded increased levels of vibration during construction activity outside the building. During the construction period, vasodilator responses to acetylcholine and SNP were abolished, and MaxiK single-channel current opening frequency and open-state probability in cell-attached patches of isolated MCA myocytes were dramatically decreased. Recovery of acetylcholine- and SNP-induced dilation was minimal in MCA from rats studied after completion of construction but housed in the animal facility during construction, whereas responses to acetylcholine and SNP were intact in rats purchased, housed, and studied after construction. Baseline levels of vibration returned after the completion of construction, concomitant with the recovery of normal endothelium-dependent vasodilation to acetylcholine and of NO sensitivity assessed by using SNP in MCA from animals obtained after construction. The results of this study indicate that the vibration associated with nearby construction can have highly disruptive effects on crucial physiologic phenotypes.

Noninvasive Imaging and Correlative Histology of Cone Photoreceptor Structure in the Pig Retina.

PURPOSE: To evaluate different methods of studying cone photoreceptor structure in wild-type (WT) and transgenic pigs carrying the human rhodopsin P23H mutant gene (TgP23H). METHODS: For in vivo imaging, pigs were anesthetized with tiletamine-zolazepam and isoflurane and given lidocaine-bupivacaine retrobulbar injections. Stay sutures and a custom head mount were used to hold and steer the head for adaptive optics scanning light ophthalmoscopy (AOSLO). Six WT and TgP23H littermates were imaged at postnatal day 30 (P30), P90, and P180 with AOSLO and optical coherence tomography (OCT), and two additional sets of littermates were imaged at P3 and P15 with OCT only. AOSLO imaging and correlative differential interference contrast microscopy were performed on a P240 WT pig and on WT and TgP23H littermates at P30 and P180. RESULTS: AOSLO cone density generally underestimates histology density (mean difference ± SD = 24.8% ± 21.4%). The intensity of the outer retinal hyperreflective OCT band attributed to photoreceptors is attenuated in TgP23H pigs at all ages. In contrast, AOSLO images show cones that retain inner and outer segments through P180. At retinal locations outside the visual streak, TgP23H pigs show a heterogeneous degenerating cone mosaic by using two criteria: variable contrast on a split detector AOSLO and high reflectivity on a confocal AOSLO. CONCLUSIONS: AOSLO reveals that the cone mosaic is similar to ex vivo histology. Its use as a noninvasive tool will enable observation of morphologic changes that arise in the cone mosaic of TgP23H pigs over time. TRANSLATIONAL RELEVANCE: Pigs are widely used for translational studies, and the ability to noninvasively assess cellular changes in the cone mosaic will facilitate more detailed investigations of new retinal disease models as well as outcomes of potential therapies.

Noninvasive imaging of the tree shrew eye: Wavefront analysis and retinal imaging with correlative histology.

Tree shrews are small mammals with excellent vision and are closely related to primates. They have been used extensively as a model for studying refractive development, myopia, and central visual processing and are becoming an important model for vision research. Their cone dominant retina (∼95% cones) provides a potential avenue to create new damage/disease models of human macular pathology and to monitor progression or treatment response. To continue the development of the tree shrew as an animal model, we provide here the first measurements of higher order aberrations along with adaptive optics scanning light ophthalmoscopy (AOSLO) images of the photoreceptor mosaic in the tree shrew retina. To compare intra-animal in vivo and ex vivo cone density measurements, the AOSLO images were matched to whole-mount immunofluorescence microscopy. Analysis of the tree shrew wavefront indicated that the optics are well-matched to the sampling of the cone mosaic and is consistent with the suggestion that juvenile tree shrews are nearly emmetropic (slightly hyperopic). Compared with in vivo measurements, consistently higher cone density was measured ex vivo, likely due to tissue shrinkage during histological processing. Tree shrews also possess massive mitochondria ("megamitochondria") in their cone inner segments, providing a natural model to assess how mitochondrial size affects in vivo retinal imagery. Intra-animal in vivo and ex vivo axial distance measurements were made in the outer retina with optical coherence tomography (OCT) and transmission electron microscopy (TEM), respectively, to determine the origin of sub-cellular cone reflectivity seen on OCT. These results demonstrate that these megamitochondria create an additional hyper-reflective outer retinal reflective band in OCT images. The ability to use noninvasive retinal imaging in tree shrews supports development of this species as a model of cone disorders.

Evaluating seasonal changes of cone photoreceptor structure in the 13-lined ground squirrel.

Cone photoreceptors of the 13-lined ground squirrel (13-LGS) undergo reversible structural changes during hibernation, including cone outer segment disc degeneration and inner segment mitochondria depletion. Here, we evaluated cone structure with adaptive optics scanning light ophthalmoscopy (AOSLO) before, during, and after hibernation. Also, intra-animal comparisons of cone structure were made at distinct physiological states (pre-hibernation, torpor, interbout euthermia, and post-hibernation) with AOSLO and transmission electron microscopy. Our results indicate that the 13-LGS cone mosaic is only transiently affected by structural remodeling during hibernation. Outer segment remodeling starts during torpid states during a period of fall transition in room temperature, with more severe structural changes during bouts of torpor in cold temperature. Cones return to euthermic-like structure during brief periods of interbout euthermia and recover normal waveguiding properties as soon as 24 h post-hibernation. Cone structure is visible with split-detector AOSLO throughout hibernation, providing evidence that intact outer segments are not necessary to visualize cones with this technique. Despite the changes to cone structure during hibernation, cone density and packing remained unchanged throughout the seasonal cycle. Pairing non-invasive imaging with ultrastructural assessment may provide insight to the biological origins of cone photoreceptor signals observed with AOSLO.

Platelet Gene Therapy Promotes Targeted Peripheral Tolerance by Clonal Deletion and Induction of Antigen-Specific Regulatory T Cells.

Delivery of gene therapy as well as of biologic therapeutics is often hampered by the immune response of the subject receiving the therapy. We have reported that effective gene therapy for hemophilia utilizing platelets as a delivery vehicle engenders profound tolerance to the therapeutic product. In this study, we investigated whether this strategy can be applied to induce immune tolerance to a non-coagulant protein and explored the fundamental mechanism of immune tolerance induced by platelet-targeted gene delivery. We used ovalbumin (OVA) as a surrogate non-coagulant protein and constructed a lentiviral vector in which OVA is driven by the platelet-specific αIIb promoter. Platelet-specific OVA expression was introduced by bone marrow transduction and transplantation. Greater than 95% of OVA was stored in platelet α-granules. Control mice immunized with OVA generated OVA-specific IgG antibodies; however, mice expressing OVA in platelets did not. Furthermore, OVA expression in platelets was sufficient to prevent the rejection of skin grafts from CAG-OVA mice, demonstrating that immune tolerance developed in platelet-specific OVA-transduced recipients. To assess the mechanism(s) involved in this tolerance we used OTII mice that express CD4+ effector T cells specific for an OVA-derived peptide. After platelet-specific OVA gene transfer, these mice showed normal thymic maturation of the T cells ruling against central tolerance. In the periphery, tolerance involved elimination of OVA-specific CD4+ effector T cells by apoptosis and expansion of an OVA-specific regulatory T cell population. These experiments reveal the existence of natural peripheral tolerance processes to platelet granule contents which can be co-opted to deliver therapeutically important products.

Treatment with ActRIIB-mFc Produces Myofiber Growth and Improves Lifespan in the Acta1 H40Y Murine Model of Nemaline Myopathy.

Nemaline myopathies (NMs) are a group of congenital muscle diseases caused by mutations in at least 10 genes and associated with a range of clinical symptoms. NM is defined on muscle biopsy by the presence of cytoplasmic rod-like structures (nemaline rods) composed of cytoskeletal material. Myofiber smallness is also found in many cases of NM and may represent a cause of weakness that can be counteracted by treatment. We have used i.p. injection of activin type IIB receptor (ActRIIB)-mFc (an inhibitor of myostatin signaling) to promote hypertrophy and increase strength in our prior murine work; we therefore tested whether ActRIIB-mFc could improve weakness in NM mice through myofiber hypertrophy. We report a study of ActRIIB-mFc treatment in the Acta1 H40Y mouse model of NM. Treatment of Acta1 H40Y mice produced significant increases in body mass, muscle mass, quadriceps myofiber size, and survival, but other measurements of strength (forelimb grip strength, ex vivo measurements of contractile function) did not improve. Our studies also identified that the complications of urethral obstruction are associated with mortality in male hemizygote Acta1 H40Y mice. The incidence of urethral obstruction and histologic evidence of chronic obstruction (inflammation) were significantly lower in Acta1 H40Y mice that had been treated with ActRIIB-mFc. ActRIIB-mFc treatment produces a mild benefit to the disease phenotype in Acta1 H40Y mice.

Rat Breeding Parameters According to Floor Space Available in Cage.

The cage floor space recommended for a female rat with a litter is greater in the 8th edition of the Guide for the Care and Use of Laboratory Animals than in previous editions. As a result, research institutions using commonly available cages to house rats may not offer the recommended amount of space for a breeding pair and litter housed in the same cage. We evaluated breeding parameters in rats housed in cages with 143 in(2) (922.6 cm(2)) compared with 210 in(2) (1355 cm(2)) of floor space. Given the strains of rats typically used at our institution, a monogamous breeding pair and litter requires 164 in(2) (1058.1 cm(2)) of floor space according to the Guide. Pairs of breeding animals were housed in each type of cage; and average time between litters, number of litters born, percentage of litter weaned, numbers of pups born and weaned, and average weaning weights were evaluated. None of the breeding parameters evaluated differed according to the floor space of the cage in which the rats were housed.

PCR testing of a ventilated caging system to detect murine fur mites.

Rodents housed in microisolation caging are commonly monitored for infectious agents by the use of soiled bedding sentinels. This strategy relies on the successful transmission of rodent pathogens from the index rodents via soiled bedding to sentinel cages and the subsequent infection or colonization of sentinel rodents. When the prevalence of a pathogen is low or the target agent is not readily transmitted by soiled bedding, alternative testing methodologies should be used. Given the continued prevalence of institutions self-reporting murine fur mites and with the advent of a new sensitive and specific PCR assay for mites, we sought to determine whether the exhaust system of an individual ventilated caging (IVC) system could be used for monitoring the rack's rodent population for mites rather than relying on the responses of sentinels. We deployed single cages of mice (Mus musculus) that were known to be infested with either Radfordia affinis or Myobia musculi on a 70-cage rack, sampled the horizontal exhaust manifolds weekly, and used the new PCR assay to test these samples for mite DNA. We detected the presence of fur mites at a 94.1% probability of detection within 4 wk of placement. Therefore, we recommend swabbing and testing the shelf exhaust manifolds of IVC racks rather than relying on soiled-bedding sentinels as an indicator of the mite status of the rodents on that rack.

Comparison of 2 rat breeding schemes using conventional caging.

Compared with earlier editions, the eighth edition of the Guide for the Care and Use of Laboratory Animals recommends more cage floor space for female rats with litters. As such, conventional rat cages often do not supply the recommended floor space to maintain 2 adult rats and a litter in the same cage. We evaluated 2 breeding schemes using traditional cages that afford 140 in.(2) (903 cm(2)) of floor space: (1) monogamous pairs housed continuously and (2) monogamous pairs cohoused intermittently with removal of the male rat after parturition. The results did not demonstrate a significant difference between breeding schemes in generation time, number of litters per breeding pair, percentage of litters weaned, number of pups born per breeding pair, and number of pups weaned per breeding pair. However, the average weaning weight of pups was significantly higher with scheme 1 compared with scheme 2. Collectively, these results indicate continuous housing of monogamous breeding pairs may be preferable to intermittent housing when conventional cages are used.

Comparison of methods to control floor contamination in an animal research facility.

The authors evaluated the effectiveness of adhesive mats, contamination control flooring, and shoe covers in decreasing the presence of microbial agents on animal holding room floors and footwear. Swab samples taken from animal holding room floors after the use of each product were compared with samples taken from rooms after no products were used. Swab samples were also taken from the heels and soles of the footwear of animal care staff before and after use of each product. The use of contamination control flooring or shoe covers significantly reduced the amount of organic material (as indicated by ATP levels measured by a luminometer) present on floors. Bacterial and ATP contamination of footwear was significantly lower after the use of shoe covers than after the use of adhesive mats or contamination control flooring, and the use of shoe covers led to a greater decrease in contamination before and after use than did use of either of the other two products. Although shoe covers were superior to both adhesive mats and contamination control flooring for decreasing contamination of animal room floors and footwear, facilities must take into account the contamination control standards required, the cost of the product, and the labor and time associated with product use when deciding which contamination control practices to implement.

Prematurity in mice leads to reduction in nephron number, hypertension, and proteinuria.

The nephron number at birth is a quantitative trait that correlates inversely with the risk of hypertension and chronic kidney disease later in life. During kidney development, the nephron number is controlled by multiple factors including genetic, epigenetic, and environmental modifiers. Premature birth, which represents more than 12% of annual live births in the United States, has been linked to low nephron number and the development of hypertension later in life. In this report, we describe the development of a mouse model of prematurity-induced reduction of nephron number. Premature mice, delivered 1 and 2 days early, have 17.4 ± 2.3% (n = 6) and 23.6 ± 2% (n = 10) fewer nephrons, respectively, when compared with full-term animals (12,252 ± 571 nephrons/kidney, n = 10). After 5 weeks of age, the mice delivered 2 days premature show lower real-time glomerular filtration rate (GFR, 283 ± 13 vs 389 ± 26 µL/min). The premature mice also develop hypertension (mean arterial pressure [MAP], 134 ± 18 vs 120 ± 14 mm Hg) and albuminuria (286 ± 83 vs 176 ± 59 µg albumin/mg creatinine). This mouse model provides a proof of concept that prematurity leads to reduced nephron number and hypertension, and this model will be useful in studying the pathophysiology of prematurity-induced nephron number reductions and hypertension.

Efficacy of footwear disinfection and shoe cover use in an animal research facility.

Although the amounts of money and time associated with using shoe covers or other means to prevent floor contamination in animal research facilities can be substantial, the most effective policies and practices remain unknown. In this study, the authors subjected six occupied rodent holding rooms in their animal research facility to three conditions: use of disinfectant mats; use of shoe covers; and no disinfectant mats or shoe covers. The authors took bacterial culture samples from the rooms under each condition. There was no significant difference in the mean number of colony forming units (CFUs) cultured when the disinfectant mats or shoe covers were used. However, the mean number of CFUs obtained was significantly lower when either disinfectant mats or shoe covers were used than when neither was used. These results suggest that using disinfectant mats or disposable shoe covers may reduce the bacterial load on rodent holding room floors.

The EtpA exoprotein of enterotoxigenic Escherichia coli promotes intestinal colonization and is a protective antigen in an experimental model of murine infection.

The enterotoxigenic Escherichia coli (ETEC) strains are major causes of morbidity and mortality due to diarrheal illness in developing countries. At present, there is no broadly protective vaccine for this diverse group of pathogens. The EtpA protein, identified in ETEC H10407 in a recent search for candidate immunogens, is a large glycosylated exoprotein secreted via two-partner secretion (TPS). Similar to structurally related molecules, EtpA functions in vitro as an adhesin. The studies reported here use a recently developed murine model of ETEC intestinal colonization to examine the immunogenicity and protective efficacy of EtpA. We report that mice repeatedly exposed to ETEC are protected from subsequent colonization and that they mount immune responses to both EtpA and its presumed two-partner secretion transporter (EtpB) during the course of experimental infection. Furthermore, isogenic etpA deletion mutants were impaired in the colonization of mice, and intranasal immunization of mice with recombinant EtpA conferred protection against ETEC H10407 in this model. Together, these data suggest that EtpA is required for optimal colonization of the intestine, findings paralleling those of previous in vitro studies demonstrating its role in adherence. EtpA and other TPS proteins may be viable targets for ETEC vaccine development.

Importance of heat-labile enterotoxin in colonization of the adult mouse small intestine by human enterotoxigenic Escherichia coli strains.

Enterotoxigenic Escherichia coli (ETEC) infections are a significant cause of diarrheal disease and infant mortality in developing countries. Studies of ETEC pathogenesis relevant to vaccine development have been greatly hampered by the lack of a suitable small-animal model of infection with human ETEC strains. Here, we demonstrate that adult immunocompetent outbred mice can be effectively colonized with the prototypical human ETEC H10407 strain (colonization factor antigen I; heat-labile and heat-stable enterotoxin positive) and that production of heat-labile holotoxin provides a significant advantage in colonization of the small intestine in this model.

Toxic epidermal necrolysis in two rhesus macaques (Macaca mulatta) after administration of rituximab.

A 2- and a 7-year-old rhesus macaque developed toxic epidermal necrolysis (TEN) after administration of ritux imab. Rituximab, a chimeric monoclonal antibody (mAb) directed against the CD20 antigen on B lymphocytes, is used to treat certain B cell neoplasias. The macaques were part of a gene therapy study that involved administering an adeno-associated viral vector encoding human factor IX (hFIX) to the animals. Both animals developed antibody against hFIX, which eliminated expression of the protein. Rituximab was administered to deplete the population of B cells producing antibodies against the protein. Two days after treatment, the 7-year-old animal developed erythemic skin lesions that rapidly progressed in severity, resulting in epidermal sloughing and ulceration. Despite aggressive treatment with analgesics, antibiotics, and corticosteroids, the animal had to be euthanized 5 days later. The 2-year-old macaque had no reaction to the initial dose of rituximab and received a second infusion 2 weeks later. Two days after drug administration, skin lesions developed; aggressive analgesic, antibiotic, and corticosteroid treatment was initiated, and the lesions resolved. A third rituximab dose was given approximately 2 months after the second. Skin lesions developed and were treated. The animal made a full recovery. In both cases, skin biopsies were taken and histopathologic findings were consistent with TEN. A severe, life-threatening condition, TEN manifests as an intolerance reaction in the skin. The most common cause of TEN is a response to previous drug administration. To our knowledge, this condition has not been reported in association with rituximab administration in macaques.