Quantitative performance assessment of Ultivue multiplex panels in formalin-fixed, paraffin-embedded human and murine tumor specimens.

We present a rigorous validation strategy to evaluate the performance of Ultivue multiplex immunofluorescence panels. We have quantified the accuracy and precision of four different multiplex panels (three human and one mouse) in tumor specimens with varying levels of T cell density. Our results show that Ultivue panels are typically accurate wherein the relative difference in cell proportion between a multiplex image and a 1-plex image is less than 20% for a given biomarker. Ultivue panels exhibited relatively high intra-run precision (CV ≤ 25%) and relatively low inter-run precision (CV >> 25%) which can be remedied by using local intensity thresholding to gate biomarker positivity. We also evaluated the reproducibility of cell-cell distance estimates measured from multiplex images which show high intra- and inter-run precision. We introduce a new metric, multiplex labeling efficiency, which can be used to benchmark the overall fidelity of the multiplex data across multiple batch runs. Taken together our results provide a comprehensive characterization of Ultivue panels and offer practical guidelines for analyzing multiplex images.

Characterizing Intra-Tumor and Inter-Tumor Variability of Immune Cell Infiltrates in Murine Syngeneic Tumors.

Murine tumors are indispensable model systems in preclinical immuno-oncology research. While immunologic heterogeneity is well-known to be an important factor that can influence treatment outcome, there is a severe paucity of data concerning the nature of this heterogeneity in murine tumor models. Using serial sectioning methodology combined with IHC analysis and whole-slide image analysis, the depth-dependent variation in immune-cell abundance in tumor specimens was investigated at single-cell resolution. Specifically, intra- and intertumor variability in cell density of nine immune-cell biomarkers was quantified in multiple murine tumor models. The analysis showed that intertumor variability was typically the dominant source of variation in measurements of immune-cell densities. Statistical power analysis revealed the effect of group size and variance in immune-cell density on the predictive power of detecting a statistically meaningful fold-change in immune-cell density. Intertumor variability in the ratio of immune-cell densities showed distinct patterns in select tumor models and revealed the existence of strong correlations between select biomarker pairs. Furthermore, the relative proportion of immune cells at different depths across tumor samples was preserved in some but not all tumor models, thereby revealing the existence of compositional heterogeneity. Taken together, these results reveal novel insights into the nature of immunologic heterogeneity, which is not accessible through typical omics approaches.

An orthogonal methods assessment of topical drug concentrations in skin and the impact for risk assessment in the viable epidermis.

Systemic toxicity assessments for oral or parenteral drugs often utilize the concentration of drug in plasma to enable safety margin calculations for human risk assessment. For topical drugs, there is no standard method for measuring drug concentrations in the stratum basale of the viable epidermis. This is particularly important since the superficial part of the epidermis, the stratum corneum (SC), is nonviable and where most of a topically applied drug remains, never penetrating deeper into the skin. We investigated the relative concentrations of a prototype kinase inhibitor using punch biopsy, laser capture microdissection, and imaging mass spectrometry methods in the SC, stratum basale, and dermis of minipig skin following topical application as a cream formulation. The results highlight the value of laser capture microdissection and mass spectrometry imaging in quantifying the large difference in drug concentration across the skin and even within the epidermis, and supports use of these methods for threshold-based toxicity risk assessments in specific anatomic locations of the skin, like of the stratum basale.

Comparative nonclinical assessments of the biosimilar PF-06410293 and originator adalimumab.

Adalimumab, a recombinant fully human monoclonal antibody targeting tumor necrosis factor (TNF), is approved in the United States and Europe to treat various inflammatory and autoimmune indications. Biosimilars are approved biologics highly similar, but not identical, to approved biotherapeutics. To support clinical development of PF-06410293, an adalimumab biosimilar, nonclinical studies evaluated the structural, functional, toxicologic, and toxicokinetic similarity to originator adalimumab sourced from the United States (adalimumab-US) and European Union (adalimumab-EU). Structural similarity was assessed by peptide mapping. Biologic activity was measured via inhibition of TNF-induced apoptosis and Fc-based functionality assessments. In vivo nonclinical similarity was evaluated in a toxicity study in cynomolgus monkeys administered subcutaneous PF-06410293 or adalimumab-EU (0 or 157 mg/kg/week). Peptide mapping demonstrated PF-06410293, adalimumab-US, and adalimumab-EU had identical amino acid sequences. Comparative functional and binding assessments were similar. Effects of PF-06410293 and adalimumab-EU were similar and limited to pharmacologically mediated decreased cellularity of lymphoid follicles and germinal centers in spleen. Toxicokinetics were similar; maximum plasma concentration and area-under-the-concentration-time curve ratio of PF-06410293:adalimumab-EU ranged from 1.0 to 1.2. These studies supported PF-06410293 entry into clinical development. Many regulatory agencies now only request nonclinical in vivo testing if there is residual uncertainty regarding biosimilarity after in vitro analytical studies.

Quantitative biodistribution of biotherapeutics at whole body, organ and cellular levels by autoradiography.

AIM: Tools for mapping and quantifying monoclonal antibody (mAb) and peptide biotherapeutics distribumtion were evaluated by comparing data from three independent methods conducted at the whole body, organ or tissue, and cellular levels. MATERIALS & METHODS: [3H]-mAb1 and [3H]-peptide A were administered intravenously to rats followed by quantitative whole-body autoradiography, kidney macro-autoradiography and micro-autoradiography. RESULTS: [3H]-mAb1 and [3H]-peptide A concentrations were measured in anatomical regions ranging from whole body to whole organ to sub-organ level, such as the kidney glomerulus, with increasing resolution. The tissue/blood [3H]-mAb1 concentrations in selected kidney microenvironments were comparable among the three quantitative methods. CONCLUSION: Quantitative whole-body autoradiography, tissue macro-autoradiography and micro-autoradiography all provide useful tools for quantifying the concentrations of biotherapeutics at different anatomical levels in tissues, facilitating better predictions of efficacy and toxicity.

Accidental and Programmed Cell Death in Investigative and Toxicologic Pathology.

Cellular development and homeostasis are regulated via programmed cell death (PCD; apoptosis), which is a genetically regulated cellular process. Accidental cell death (ACD; necrosis) can be triggered by chemical, physical, or mechanical stress. Necrosis is the presence of dead tissues or cells in a living organism regardless of the initiating process and can be observed in infectious and non-infectious diseases and toxicities. This article describes tissue-based immunohistotechnical protocols used for assessing PCD and necrosis in formalin-fixed tissues obtained from preclinical species used in investigative and toxicologic pathology. Two commonly employed protocols for the identification of PCD and necrosis are described in this article: immunohistochemistry (IHC) for cleaved caspase 3, and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL). TUNEL has been used to detect DNA fragmentation by labeling the terminal ends of nucleic acids in necrotic and apoptotic cells. © 2018 by John Wiley & Sons, Inc.

Soluble Fn14 Is Detected and Elevated in Mouse and Human Kidney Disease.

The cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are upregulated in tissue injury to mediate local tissue responses including inflammation and tissue remodeling. We found that in various models of kidney disease, Fn14 expression (mRNA and protein) is upregulated in the kidney. These models include: lupus nephritis mouse models (Nephrotoxic serum Transfer Nephritis and MRL.Faslpr/lpr), acute kidney injury models (Ischemia reperfusion injury and Folic acid injury), and a ZSF-1 diabetic nephropathy rat model. Fn14 expression levels correlate with disease severity as measured by disease histology. We have also shown for the first time the detection of soluble Fn14 (sFn14) in the urine and serum of mice. Importantly, we found the sFn14 levels are markedly increased in the diseased mice and are correlated with disease biomarkers including proteinuria and MCP-1. We have also detected sFn14 in human plasma and urine. Moreover, sFn14 levels, in urine are significantly increased in DN patients and correlated with proteinuria and MCP-1 levels. Thus our data not only confirm the up-regulation of Fn14/TWEAK pathway in kidney diseases, but also suggest a novel mechanism for its regulation by the generation of sFn14. The correlation of sFn14 levels and disease severity suggest that sFn14 may serve as a potential biomarker for both acute and chronic kidney diseases.

Tofacitinib attenuates pathologic immune pathways in patients with psoriasis: A randomized phase 2 study.

BACKGROUND: Tofacitinib is an oral Janus kinase inhibitor being investigated for psoriasis. OBJECTIVE: We sought to elucidate the molecular mechanisms underlying the clinical efficacy of tofacitinib in patients with psoriasis. METHODS: Twelve patients with plaque psoriasis were randomized (3:1) to receive 10 mg of tofacitinib or placebo twice daily for 12 weeks. Biopsy specimens were taken from nonlesional (baseline) and lesional (baseline, days 1 and 3, and weeks 1, 2, 4, and 12) skin. Biopsy specimens were examined for psoriatic epidermal features (thickness, Ki67(+) keratinocytes and keratin 16 [KRT16] mRNA expression, and phosphorylated signal transducer and activator of transcription [pSTAT](+) nuclei) and T-cell and dendritic cell (DC) subsets by using immunohistochemistry, and mRNA transcripts were quantified by using a microarray. RESULTS: In lesional skin keratinocyte pSTAT1 and pSTAT3 staining was increased at baseline but reduced after 1 day of tofacitinib (baseline, median of 1290 pSTAT1(+) cells/µm(2); day 1, median of 332 pSTAT1(+) cells/µm(2); and nonlesional, median of 155 pSTAT1(+) cells/µm(2)). Epidermal thickness and KRT16 mRNA expression were significantly and progressively reduced after days 1 and 3 of tofacitinib administration, respectively (eg, KRT16 decreased 2.74-fold, day 3 vs baseline, P = .016). Decreases in DC and T-cell numbers were observed after weeks 1 and 2, respectively. At week 4, significant decreases in IL-23/TH17 pathways were observed that persisted through week 12. Improvements in clinical and histologic features were strongly associated with changes in expression of psoriasis-related genes and reduction in IL-17 gene expression. CONCLUSIONS: Tofacitinib has a multitiered response in patients with psoriasis: (1) rapid attenuation of keratinocyte Janus kinase/STAT signaling; (2) removal of keratinocyte-induced cytokine signaling, leading to reductions in pathologic DC and T-cell numbers to nonlesional levels; and (3) inhibition of the IL-23/TH17 pathway.

Understanding Lung Deposition of Alpha-1 Antitrypsin in Acute Experimental Mouse Lung Injury Model Using Fluorescence Microscopy.

Human plasma-derived α1-antitrypsin (AAT) delivered by intravenous infusion is used as augmentation therapy in patients with emphysema who have a genetic mutation resulting in deficiency of AAT. Inhalation is an alternative route of administration that can potentially increase the efficacy and convenience of treatment. This study was conducted to determine whether delivery to the lungs, initially via the intratracheal (IT) route of administration, would deliver efficacious levels of a recombinant AAT (rAAT) to the site of action in the lungs in mice. 125I-radiolabeled rAAT, fluorophore-conjugated rAAT (rAAT-Alexa488), and NE680 (neutrophil elastase 680, a silent fluorescent substrate of neutrophil elastase which fluoresces in the near-infrared range upon activation by neutrophil elastase) were used to characterize the pharmacokinetics and tissue distribution profile, distribution of rAAT within the lung, and efficacy of rAAT to inhibit neutrophil elastase at the site of action, respectively. The study has demonstrated that rAAT was able to gain access to locations where neutrophil elastase was localized. The histochemical quantification of rAAT activity relative to dose at the site of action provided here will improve confidence in predicting the human dose via the inhalation route.

Identification of Promising Urinary MicroRNA Biomarkers in Two Rat Models of Glomerular Injury.

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate protein levels posttranscriptionally. miRNAs play important regulatory roles in many cellular processes and have been implicated in several diseases. Recent studies have reported significant levels of miRNAs in a variety of body fluids, raising the possibility that miRNAs could serve as useful biomarkers. Here, changes in miRNA expression patterns are described in 2 different rodent models of glomerular injury (acute puromycin aminonucleoside nephropathy and passive Heymann nephritis). By employing 2 different modes of glomerular insult, oxidative stress and immune-mediated toxicity, miRNA changes in both isolated glomeruli as well as urine specimens allow for identification of urinary miRNA biomarkers that are suggestive of drug-induced injury specifically to the glomerulus. Subsets of glomerular urinary miRNAs associated with these different modes of glomerular toxicity seem to be dependent on the mechanism of the induced injury, while 9 miRNAs that changed early in both glomerular and urine specimens were common to both studies. We further show that the miRNAs identified as mechanism-specific early glomerular injury biomarkers target key pathways and transcripts relevant to the type of insult, while the insult-independent changes might serve as ideal glomerular injury biomarkers.

Identification of tubular injury microRNA biomarkers in urine: comparison of next-generation sequencing and qPCR-based profiling platforms.

BACKGROUND: MicroRNAs (miRNAs) are small, non-coding RNAs that regulate protein levels post-transcriptionally. miRNAs play important regulatory roles in many cellular processes and have been implicated in several diseases. Recent studies have reported significant levels of miRNAs in a variety of body fluids, raising the possibility that miRNAs could serve as useful biomarkers. Next-generation sequencing (NGS) is increasingly employed in biomedical investigations. Although concordance between this platform and qRT-PCR based assays has been reported in high quality specimens, information is lacking on comparisons in biofluids especially urine. Here we describe the changes in miRNA expression patterns in a rodent model of renal tubular injury (gentamicin). Our aim is to compare RNA sequencing and qPCR based miRNA profiling in urine specimen from control and rats with confirmed tubular injury. RESULTS: Our preliminary examination of the concordance between miRNA-seq and qRT-PCR in urine specimen suggests minimal agreement between platforms probably due to the differences in sensitivity. Our results suggest that although miRNA-seq has superior specificity, it may not detect low abundant miRNAs in urine samples. Specifically, miRNA-seq did not detect some sequences which were identified by qRT-PCR. On the other hand, the qRT-PCR analysis was not able to detect the miRNA isoforms, which made up the majority of miRNA changes detected by NGS. CONCLUSIONS: To our knowledge, this is the first time that miRNA profiling platforms including NGS have been compared in urine specimen. miRNAs identified by both platforms, let-7d, miR-203, and miR-320, may potentially serve as promising novel urinary biomarkers for drug induced renal tubular epithelial injury.

Mechanistic investigations of test article-induced pancreatic toxicity at the endocrine-exocrine interface in the rat.

Pancreatic toxicity commonly affects the endocrine or exocrine pancreas. However, it can also occur at the endocrine-exocrine interface (EEI), where the capillary network of the islet merges with the capillaries of the surrounding acinar tissue, that is, the insulo-acinar portal system. The goal of this article is to describe a novel, test article-induced pancreatic toxicity that originated at the EEI and to summarize investigations into the mechanistic basis of the injury. This injury was initially characterized by light microscopy in 7/14 day-toxicity studies in Sprague-Dawley (Crl: CD®[SD]) rats with undisclosed test articles. Microvascular injury at the interface resulted in peri-islet serum exudation, fibrin deposition, hemorrhage, inflammation, and secondary degeneration/necrosis of surrounding exocrine tissue. More chronic injury presented as islet fibrosis and lobular atrophy. Direct cytotoxicity affecting the capillary endothelium at the EEI was confirmed ultrastructurally on day 4. Endothelial microparticle and blood flow studies further confirmed endothelial involvement. Similar lesions occurred less frequently in 2 other rat strains and not in the mouse, dog, or cynomolgus macaque. In summary, in vivo and investigative study data confirmed primary endothelial cytotoxicity in the pathogenesis of this lesion and suggested that the lesion may be rat/rat strain-specific and of uncertain relevance for human safety risk assessment.

Diffusion tensor imaging detects treatment effects of FTY720 in experimental autoimmune encephalomyelitis mice.

Fingolimod (FTY720) is an orally available sphingosine-1-phosphate (S1P) receptor modulator reducing relapse frequency in patients with relapsing-remitting multiple sclerosis (RRMS). In addition to immunosuppression, neuronal protection by FTY720 has also been suggested, but remains controversial. Axial and radial diffusivities derived from in vivo diffusion tensor imaging (DTI) were employed as noninvasive biomarkers of axonal injury and demyelination to assess axonal protection by FTY720 in experimental autoimmune encephalomyelitis (EAE) mice. EAE was induced through active immunization of C57BL/6 mice using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG(35-55)). We evaluated both the prophylactic and therapeutic treatment effect of FTY720 at doses of 3 and 10 mg/kg on EAE mice by daily clinical scoring and end-point in vivo DTI. Prophylactic administration of FTY720 suppressed the disease onset and prevented axon and myelin damage when compared with EAE mice without treatment. Therapeutic treatment by FTY720 did not prevent EAE onset, but reduced disease severity, improving axial and radial diffusivity towards the control values without statistical significance. Consistent with previous findings, in vivo DTI-derived axial and radial diffusivity correlated with clinical scores in EAE mice. The results support the use of in vivo DTI as an effective outcome measure for preclinical drug development.

Acute SIV infection in sooty mangabey monkeys is characterized by rapid virus clearance from lymph nodes and absence of productive infection in germinal centers.

Lymphoid tissue immunopathology is a characteristic feature of chronic HIV/SIV infection in AIDS-susceptible species, but is absent in SIV-infected natural hosts. To investigate factors contributing to this difference, we compared germinal center development and SIV RNA distribution in peripheral lymph nodes during primary SIV infection of the natural host sooty mangabey and the non-natural host pig-tailed macaque. Although SIV-infected cells were detected in the lymph node of both species at two weeks post infection, they were confined to the lymph node paracortex in immune-competent mangabeys but were seen in both the paracortex and the germinal center of SIV-infected macaques. By six weeks post infection, SIV-infected cells were no longer detected in the lymph node of sooty mangabeys. The difference in localization and rate of disappearance of SIV-infected cells between the two species was associated with trapping of cell-free virus on follicular dendritic cells and higher numbers of germinal center CD4(+) T lymphocytes in macaques post SIV infection. Our data suggests that fundamental differences in the germinal center microenvironment prevent productive SIV infection within the lymph node germinal centers of natural hosts contributing to sustained immune competency.

Early induction of polyfunctional simian immunodeficiency virus (SIV)-specific T lymphocytes and rapid disappearance of SIV from lymph nodes of sooty mangabeys during primary infection.

Although the cellular immune response is essential for controlling SIV replication in Asian macaques, its role in maintaining nonpathogenic SIV infection in natural hosts such as sooty mangabeys (SM) remains to be defined. We have previously shown that similar to rhesus macaques (RM), SM are able to mount a T lymphocyte response against SIV infection. To investigate early control of SIV replication in natural hosts, we performed a detailed characterization of SIV-specific cellular immunity and viral control in the first 6 mo following SIV infection in SM. Detection of the initial SIV-specific IFN-γ ELISPOT response in SIVsmE041-infected SM coincided temporally with a decline in peak plasma viremia and was similar in magnitude, specificity, and breadth to SIVsmE041-infected and SIVmac239-infected RM. Despite these similarities, SM showed a greater reduction in postpeak plasma viremia and a more rapid disappearance of productively SIV-infected cells from the lymph node compared with SIVmac239-infected RM. The early Gag-specific CD8(+) T lymphocyte response was significantly more polyfunctional in SM compared with RM, and granzyme B-positive CD8(+) T lymphocytes were present at significantly higher frequencies in SM even prior to SIV infection. These findings suggest that the early SIV-specific T cell response may be an important determinant of lymphoid tissue viral clearance and absence of lymph node immunopathology in natural hosts of SIV infection.

Neurometabolite abnormalities in simian immunodeficiency virus-infected macaques with chronic morphine administration.

Opiate abuse increases the risk for human immunodeficiency virus (HIV) infection, while both opiates and HIV may impact the immune and nervous systems. To model potential interactions between opiate drugs and HIV on the brain, neurometabolite levels were evaluated in simian immunodeficiency virus (SIV)-infected macaques with or without chronic morphine administration. Over the course of the study, 58% of these SIV-infected animals progressed to acquired immune deficiency syndrome (AIDS). Brain extracts from four brain regions were evaluated with proton magnetic resonance spectroscopy. Animals with AIDS had lower N-acetyl-aspartate in all four brain regions (p ≤ 0.05) as well as lower frontal gray matter total creatine (p= 0.03), lower frontal white matter (p= 0.003) and caudate (p = 0.002) glutamate, and higher frontal white matter myo-inositol (p= 0.05) than the healthier non-AIDS macaques. Morphine-dependent animals had higher levels of myo-inositol in the putamen (p = 0.003), especially those with AIDS. In the animals with AIDS, those with morphine dependence had higher total creatine in the frontal white matter (p= 0.04) than those treated with saline, which in turn had lower creatine than saline-injected animals without AIDS (p = 0.04), leading to an interaction between the effects of morphine and AIDS on total creatine in this brain region (ANOVA p = 0.02). The majority of these brain metabolites correlated with viral counts indicating more severe metabolite abnormalities in animals with higher viral loads or set points. Collectively, these findings suggest that chronic morphine may protect against the neurotoxic effect of AIDS and reinforce the importance of maintaining a low viral load in AIDS.

Cloning and characterization of rhesus IL-18 binding protein, a natural antagonist to IL-18.

IL-18 is a proinflammatory cytokine that is important for host defense, but is also involved in the pathogenesis of a number of disease processes, ranging from autoimmune disorders to atherosclerosis. IL-18 binding protein (IL-18BP) is a constitutively expressed glycoprotein that specifically neutralizes the effects of IL-18, resulting in decreased production of IFN-gamma and reduction in Th1 immune responses. In this study we cloned and sequenced a full-length cDNA of the rhesus IL-18BP (RhIL-18BP) from the spleen of rhesus macaques (Macaca mulatta) and compared its nucleotide and amino acid sequences to the functional murine and human IL-18BP orthologues. In addition, we fused RhIL-18BP to the Fc portion of human IgG1 to make recombinant RhIL-18BP x Fcgamma1 in order to facilitate its detection by Western blot analysis and determined the approximate molecular weight of RhIL-18BP x Fcgamma1 to be 66 kD. With this fusion protein, we showed that RhIL-18BP was functional and could significantly reduce murine IL-18 and LPS-induced IFN-gamma production by murine splenocytes. Furthermore, we demonstrated the expression of IL-18BP in atherosclerotic lesions in a rhesus model of atherosclerosis, underscoring the need to fully understand the role of this protein as a primary negative regulator of IL-18 in multiple disease processes.

Impact of short-term combined antiretroviral therapy on brain virus burden in simian immunodeficiency virus-infected and CD8+ lymphocyte-depleted rhesus macaques.

Antiretroviral drugs suppress virus burden in the cerebrospinal fluid of HIV-infected individuals; however, the direct effect of antiretrovirals on virus replication in brain parenchyma is poorly understood. We investigated the effect of short-term combined antiretroviral therapy (CART) on brain virus burden in rhesus monkeys using the CD8-depletion model of accelerated simian immunodeficiency virus (SIV) encephalitis. Four monkeys received CART (consisting of the nonpenetrating agents PMPA and RCV) for four weeks, beginning 28 days after SIV inoculation. Lower virus burdens were measured by real-time RT-PCR in four of four regions of brain from monkeys that received CART as compared with four SIV-infected, untreated controls; however, the difference was only significant for the frontal cortex (P < 0.05). In contrast, significantly lower virus burdens were measured in plasma and four of five lymphoid compartments from animals that received CART. Surprisingly, despite normalization of neuronal function in treated animals, the numbers of activated macrophages/microglia and the magnitude of TNF-alpha mRNA expression in brain were similar between treated animals and controls. These results suggest that short-term therapy with antiretrovirals that fail to penetrate the blood-cerebrospinal fluid barrier can reduce brain virus burden provided systemic virus burden is suppressed; however, longer treatment may be required to completely resolve encephalitic lesions and microglial activation, which may reflect the longer half-life of the principal target cells of HIV/SIV in the brain (macrophages) versus lymphoid tissues (T lymphocytes).

Acute lymphoid and gastrointestinal toxicity induced by selective p38alpha map kinase and map kinase-activated protein kinase-2 (MK2) inhibitors in the dog.

Exposure to moderately selective p38alpha mitogen-activated protein kinase (MAPK) inhibitors in the Beagle dog results in an acute toxicity consisting of mild clinical signs (decreased activity, diarrhea, and fever), lymphoid necrosis and depletion in the gut-associated lymphoid tissue (GALT), mesenteric lymph nodes and spleen, and linear colonic and cecal mucosal hemorrhages. Lymphocyte apoptosis and necrosis in the GALT is the earliest and most prominent histopathologic change observed, followed temporally by neutrophilic infiltration and acute inflammation of the lymph nodes and spleen and multifocal mucosal epithelial necrosis and linear hemorrhages in the colon and cecum. These effects are not observed in the mouse, rat, or cynomolgus monkey. To further characterize the acute toxicity in the dog, a series of in vivo, in vitro, and immunohistochemical studies were conducted to determine the relationship between the lymphoid and gastrointestinal (GI) toxicity and p38 MAPK inhibition. Results of these studies demonstrate a direct correlation between p38alpha MAPK inhibition and the acute lymphoid and gastrointestinal toxicity in the dog. Similar effects were observed following exposure to inhibitors of MAPK-activated protein kinase-2 (MK2), further implicating the role of p38alpha MAPK signaling pathway inhibition in these effects. Based on these findings, the authors conclude that p38alpha MAPK inhibition results in acute lymphoid and GI toxicity in the dog and is unique among the species evaluated in these studies.

Relative transmissibility of an R5 clade C simian-human immunodeficiency virus across different mucosae in macaques parallels the relative risks of sexual HIV-1 transmission in humans via different routes.

BACKGROUND: Worldwide, approximately 90% of all human immunodeficiency virus (HIV) transmissions occur mucosally; almost all involve R5 strains. Risks of sexual HIV acquisition are highest for rectal, then vaginal, and finally oral exposures. METHODS: Mucosal lacerations may affect the rank order of susceptibility to HIV but cannot be assessed in humans. We measured relative virus transmissibility across intact mucosae in macaques using a single stock of SHIV-1157ipd3N4, a simian-human immunodeficiency virus encoding a primary R5 HIV clade C env (SHIV-C). RESULTS: The penetrability of rhesus macaque mucosae differed significantly, with rectal challenge requiring the least virus, followed by vaginal and then oral routes (P = .031, oral vs vaginal; P < .001 rectal vs vaginal). These findings imply that intrinsic mucosal properties are responsible for the differential mucosal permeability. The latter paralleled the rank order reported for humans, with relative risk estimates within the range of epidemiological human studies. To test whether inflammation facilitates virus transmission--as predicted from human studies--we established a macaque model of localized buccal inflammation. Systemic infection occurred across inflamed but not normal buccal mucosa. CONCLUSION: Our primate data recapitulate virus transmission risks observed in humans, thus establishing R5 SHIV-1157ipd3N4 in macaques as a robust model system to study cofactors involved in human mucosal HIV transmission and its prevention.