Utility of CD138/syndecan-1 immunohistochemistry for localization of plasmacytes is tissue-dependent in B6 mice.

OBJECTIVE: Inflammation is present in many diseases and identification of immune cell infiltration is a common assessment. CD138 (syndecan-1) is a recommended immunohistochemical marker for human plasmacytes although it is also expressed in various epithelia and tumors. Similarly, CD138 is a marker for murine plasmacytes, but its tissue immunostaining is not well-defined. Endogenous CD138 expression is an important confounding factor when evaluating plasmacyte infiltration. We studied two plasmacyte markers (CD138 and Kappa light chains) for endogenous immunostaining in five organs and one tumor from B6 mice. RESULTS: Plasmacytes in Peyer's patches were positive for CD138 and Kappa markers without endogenous immunostaining. Endogenous CD138 immunostaining was widespread in liver, kidney, lung and a malignant peripheral nerve sheath tumor (MPNST) versus regionalized immunostaining in skin and small intestine wall. Endogenous Kappa immunostaining was absent in all tissues except for plasmacytes. Tissues with widespread endogenous CD138 immunostaining were contrasted by absence of endogenous Kappa immunostaining. Here, plasmacytes would not be distinguished by CD138, but would be obvious by Kappa immunostaining. Our study suggests that utility of immunostaining for plasmacytes by CD138 is tissue dependent in mice. Additionally, Kappa immunostaining may be a useful alternative in mouse tissues with confounding endogenous CD138 immunostaining.

Lack of airway submucosal glands impairs respiratory host defenses.

Submucosal glands (SMGs) are a prominent structure that lines human cartilaginous airways. Although it has been assumed that SMGs contribute to respiratory defense, that hypothesis has gone without a direct test. Therefore, we studied pigs, which have lungs like humans, and disrupted the gene for ectodysplasin (EDA-KO), which initiates SMG development. EDA-KO pigs lacked SMGs throughout the airways. Their airway surface liquid had a reduced ability to kill bacteria, consistent with SMG production of antimicrobials. In wild-type pigs, SMGs secrete mucus that emerges onto the airway surface as strands. Lack of SMGs and mucus strands disrupted mucociliary transport in EDA-KO pigs. Consequently, EDA-KO pigs failed to eradicate a bacterial challenge in lung regions normally populated by SMGs. These in vivo and ex vivo results indicate that SMGs are required for normal antimicrobial activity and mucociliary transport, two key host defenses that protect the lung.

Glycogen depletion can increase the specificity of mucin detection in airway tissues.

OBJECTIVE: Mucin is an important parameter for detection and assessment in studies of airway disease including asthma and cystic fibrosis. Histochemical techniques are often used to evaluate mucin in tissues sections. Periodic acid Schiff (PAS) is a common technique to detect neutral mucins in tissue, but this technique also detects other tissue components including cellular glycogen. We tested whether depletion of glycogen, a common cellular constituent, could impact the detection of mucin in the surface epithelium of the trachea. RESULTS: Normal tissues stained by PAS had significantly more staining than serial sections of glycogen-depleted tissue with PAS staining (i.e. dPAS technique) based on both quantitative analysis and semiquantitative scores. Most of the excess stain by the PAS technique was detected in ciliated cells adjacent to goblet cells. We also compared normal tissues using the Alcian blue technique, which does not have reported glycogen staining, with the dPAS technique. These groups had similar amounts of staining consistent with a high degree of mucin specificity. Our results suggest that when using PAS techniques to stain airways, the dPAS approach is preferred as it enhances the specificity for airway mucin.

Allograft Inflammatory Factor 1 as an Immunohistochemical Marker for Macrophages in Multiple Tissues and Laboratory Animal Species.

Allograft inflammatory factor 1 (AIF1) is a commonly used marker for microglia in the brains of humans and some animal models but has had limited applications elsewhere. We sought to determine whether AIF1 can be used as a macrophage marker across common laboratory animal species and tissues. We studied tissues (that is, spleen, liver, and lung) with defined macrophage populations by using an AIF1 immunostaining technique previously validated in human tissue. Tissues were collected from various mouse strains (n = 20), rat strains (n = 15), pigs (n = 4), ferrets (n = 4), and humans (n = 4, lung only). All samples of liver had scattered immunostaining in interstitial cells, consistent with resident tissue macrophages (Kupffer cells). Spleen samples had cellular immunostaining of macrophages in both the red and white pulp compartments, but the red pulp had more immunostained cellular aggregates and, in some species, increased immunostaining intensity compared with white pulp. In lung, alveolar macrophages had weak to moderate staining, whereas interstitial and perivascular macrophages demonstrated moderate to robust staining. Incidental lesions and tissue changes were detected in some sections, including a tumor, inducible bronchus-associated lymphoid tissue, and inflammatory lesions that demonstrated AIF1 immunostaining of macrophages. Finally, we compared AIF1 immunostaining of alveolar macrophages between a hypertensive rat model (SHR strain) and a normotensive model (WKY strain). SHR lungs had altered intensity and distribution of immunostaining in activated macrophages compared with macrophages of WKY lungs. Overall, AIF1 immunostaining demonstrated reproducible macrophage staining across multiple species and tissue types. Given the increasing breadth of model species used to study human disease, the use of cross-species markers and techniques can reduce some of the inherent variability within translational research.

Immunohistochemical Markers for Prospective Studies in Neurofibromatosis-1 Porcine Models.

Neurofibromatosis type 1 (NF1) is a common, cancer-predisposing disease caused by mutations in the NF1 tumor gene. Patients with NF1 have an increased risk for benign and malignant tumors of the nervous system (e.g., neurofibromas, malignant peripheral nerve sheath tumors, gliomas) and other tissues (e.g., leukemias, rhabdomyosarcoma, etc.) as well as increased susceptibility to learning disabilities, chronic pain/migraines, hypertension, pigmentary changes, and developmental lesions (e.g., tibial pseudoarthrosis). Pigs are an attractive and upcoming animal model for future NF1 studies, but a potential limitation to porcine model research has been the lack of validated reagents for direct translational study to humans. To address that issue, we used formalin-fixed tissues (human and pigs) to evaluate select immunohistochemical markers (activated caspase-3, allograft inflammatory factor-1, beta-tubulin III, calbindin D, CD13, CD20, desmin, epithelial membrane antigen, glial fibrillary acidic protein, glucose transporter-1, laminin, myelin basic protein, myoglobin, proliferating cell nuclear antigen, S100, vimentin, and von Willebrand factor). The markers were validated by comparing known expression and localization in human and pig tissues. Validation of these markers on fixed tissues will facilitate prospective immunohistochemical studies of NF1 pigs, as well as other pig models, in a more efficient, reproducible, and translationally relevant manner.

A method for histopathological study of the multifocal nature of spinal cord lesions in murine experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a well-established mouse model for multiple sclerosis and is characterized by infiltration of mononuclear cells and demyelination within the central nervous system along with the clinical symptoms of paralysis. EAE is a multifocal and random disease, which sometimes makes histopathologic analysis of lesions difficult as it may not be possible to predict where lesions will occur, especially when evaluating cross sections of spinal cord. Consequently, lesions may be easily missed due to limited sampling in traditional approaches. To evaluate the entire length of the spinal cord while maintaining anatomic integrity, we have developed a method to section the cord within the decalcified spinal column, which allows for the study of the multifocal nature of this disease and also minimizes handling artifact. HE and Luxol fast blue staining of these spinal cord sections revealed a paucity of lesions in some areas, while others showed marked inflammation and demyelination. The percentage of spinal cord affected by EAE was evaluated at four separate areas of longitudinally sectioned cord and it varied greatly within each animal. Immunohistochemical staining of in situ spinal cords which had undergone decalcification was successful for key immuno-markers used in EAE research including CD3 for T cells, B220 for B cells and F4/80 for murine macrophages. This method will allow investigators to look at the entire spinal cord on a single slide and evaluate the spinal cord with and without classic EAE lesions.

NIAM-deficient mice are predisposed to the development of proliferative lesions including B-cell lymphomas.

Nuclear Interactor of ARF and Mdm2 (NIAM, gene designation Tbrg1) is a largely unstudied inhibitor of cell proliferation that helps maintain chromosomal stability. It is a novel activator of the ARF-Mdm2-Tip60-p53 tumor suppressor pathway as well as other undefined pathways important for genome maintenance. To examine its predicted role as a tumor suppressor, we generated NIAM mutant (NIAM(m/m)) mice homozygous for a ß-galactosidase expressing gene-trap cassette in the endogenous gene. The mutant mice expressed significantly lower levels of NIAM protein in tissues compared to wild-type animals. Fifty percent of aged NIAM deficient mice (14 to 21 months) developed proliferative lesions, including a uterine hemangioma, pulmonary papillary adenoma, and a Harderian gland adenoma. No age-matched wild-type or NIAM(+/m) heterozygous animals developed lesions. In the spleen, NIAM(m/m) mice had prominent white pulp expansion which correlated with enhanced increased reactive lymphoid hyperplasia and evidence of systemic inflammation. Notably, 17% of NIAM mutant mice had splenic white pulp features indicating early B-cell lymphoma. This correlated with selective expansion of marginal zone B cells in the spleens of younger, tumor-free NIAM-deficient mice. Unexpectedly, basal p53 expression and activity was largely unaffected by NIAM loss in isolated splenic B cells. In sum, NIAM down-regulation in vivo results in a significant predisposition to developing benign tumors or early stage cancers. These mice represent an outstanding platform for dissecting NIAM's role in tumorigenesis and various anti-cancer pathways, including p53 signaling.

Development and translational imaging of a TP53 porcine tumorigenesis model.

Cancer is the second deadliest disease in the United States, necessitating improvements in tumor diagnosis and treatment. Current model systems of cancer are informative, but translating promising imaging approaches and therapies to clinical practice has been challenging. In particular, the lack of a large-animal model that accurately mimics human cancer has been a major barrier to the development of effective diagnostic tools along with surgical and therapeutic interventions. Here, we developed a genetically modified porcine model of cancer in which animals express a mutation in TP53 (which encodes p53) that is orthologous to one commonly found in humans (R175H in people, R167H in pigs). TP53(R167H/R167H) mutant pigs primarily developed lymphomas and osteogenic tumors, recapitulating the tumor types observed in mice and humans expressing orthologous TP53 mutant alleles. CT and MRI imaging data effectively detected developing tumors, which were validated by histopathological evaluation after necropsy. Molecular genetic analyses confirmed that these animals expressed the R167H mutant p53, and evaluation of tumors revealed characteristic chromosomal instability. Together, these results demonstrated that TP53(R167H/R167H) pigs represent a large-animal tumor model that replicates the human condition. Our data further suggest that this model will be uniquely suited for developing clinically relevant, noninvasive imaging approaches to facilitate earlier detection, diagnosis, and treatment of human cancers.

Gastrointestinal pathology in juvenile and adult CFTR-knockout ferrets.

Cystic fibrosis (CF) is a multiorgan disease caused by loss of a functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in many epithelia of the body. Here we report the pathology observed in the gastrointestinal organs of juvenile to adult CFTR-knockout ferrets. CF gastrointestinal manifestations included gastric ulceration, intestinal bacterial overgrowth with villous atrophy, and rectal prolapse. Metagenomic phylogenetic analysis of fecal microbiota by deep sequencing revealed considerable genotype-independent microbial diversity between animals, with the majority of taxa overlapping between CF and non-CF pairs. CF hepatic manifestations were variable, but included steatosis, necrosis, biliary hyperplasia, and biliary fibrosis. Gallbladder cystic mucosal hyperplasia was commonly found in 67% of CF animals. The majority of CF animals (85%) had pancreatic abnormalities, including extensive fibrosis, loss of exocrine pancreas, and islet disorganization. Interestingly, 2 of 13 CF animals retained predominantly normal pancreatic histology (84% to 94%) at time of death. Fecal elastase-1 levels from these CF animals were similar to non-CF controls, whereas all other CF animals evaluated were pancreatic insufficient (<2 µg elastase-1 per gram of feces). These findings suggest that genetic factors likely influence the extent of exocrine pancreas disease in CF ferrets and have implications for the etiology of pancreatic sufficiency in CF patients. In summary, these studies demonstrate that the CF ferret model develops gastrointestinal pathology similar to CF patients.

Lung phenotype of juvenile and adult cystic fibrosis transmembrane conductance regulator-knockout ferrets.

Chronic bacterial lung infections in cystic fibrosis (CF) are caused by defects in the CF transmembrane conductance regulator chloride channel. Previously, we described that newborn CF transmembrane conductance regulator-knockout ferrets rapidly develop lung infections within the first week of life. Here, we report a more slowly progressing lung bacterial colonization phenotype observed in juvenile to adult CF ferrets reared on a layered antibiotic regimen. Even on antibiotics, CF ferrets were still very susceptible to bacterial lung infection. The severity of lung histopathology ranged from mild to severe, and variably included mucus obstruction of the airways and submucosal glands, air trapping, atelectasis, bronchopneumonia, and interstitial pneumonia. In all CF lungs, significant numbers of bacteria were detected and impaired tracheal mucociliary clearance was observed. Although Streptococcus, Staphylococcus, and Enterococcus were observed most frequently in the lungs of CF animals, each animal displayed a predominant bacterial species that accounted for over 50% of the culturable bacteria, with no one bacterial taxon predominating in all animals. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry fingerprinting was used to quantify lung bacteria in 10 CF animals and demonstrated Streptococcus, Staphylococcus, Enterococcus, or Escherichia as the most abundant genera. Interestingly, there was significant overlap in the types of bacteria observed in the lung and intestine of a given CF animal, including bacterial taxa unique to the lung and gut of each CF animal analyzed. These findings demonstrate that CF ferrets develop lung disease during the juvenile and adult stages that is similar to patients with CF, and suggest that enteric bacterial flora may seed the lung of CF ferrets.

Optimal oligonucleotide sequences for TLR9 inhibitory activity in human cells: lack of correlation with TLR9 binding.

Toll-like receptor (TLR)9 performs our innate response to bacterial DNA, warning us of the presence of infection. Inhibitory oligodeoxyribonucleotides (INH-ODN) have been developed that selectively block activation of mouse TLR9. Their inhibitory motif consisting of CCx(not-C)(not-C)xxGGG (x = any base) also reduces anti-DNA antibodies in lupus mice. The current study demonstrates that this motif also provides the sequences required to block TLR9 in human B cells and human embryonic kidney (HEK) cells transfected with human TLR9. However, extending the sequence by four to five bases at the 5' end enhanced activity and this enhancement was greater when a phosphorothioate (pS) backbone replaced the native phosphodiester (pO) backbone. A series of pO-backbone INH-ODN representing a 500-fold range of activity in biologic assays was shown to cover less than a 2.5-fold range of avidity for binding human TLR9-Ig fusion protein, eliminating TLR9 ectodomain binding as the explanation for sequence-specific differences in biologic activity. With few exceptions, the relative activity of INH-ODN in Namalwa cells and HEK/human TLR9 cells was similar to that seen in mouse B cells. INH-ODN activity in human peripheral blood B cells correlated significantly with the cell line data. These results favor the conclusion that although the backbone determines strength of TLR9 binding, critical recognition of the INH-ODN sequence necessary for biologic activity is performed by a molecule that is not TLR9. These studies also identify the strongest INH-ODN for human B cells, helping to guide the selection of INH-ODN sequences for therapeutics in any situation where inflammation is enhanced by TLR9.

Aggregation and secondary loop structure of oligonucleotides do not determine their ability to inhibit TLR9.

Toll-like receptor 9 (TLR9) is an endosomal DNA sensor that warns us of the presence of infectious danger and triggers a rapid pro-inflammatory response in dendritic cells, macrophages, and B cells. The consequences of uncontrolled TLR9 activation can be detrimental for the host, contributing to the pathogenesis of bacterial septic shock or autoimmune diseases, such as systemic lupus erythematosus. Therefore, we need to develop TLR9 antagonists. We and others have created inhibitory oligonucleotides (INH-ODN) that are capable of sequence-dependent inhibition of TLR9-induced activation in both human and mouse cells. However, it is not clear whether marked differences in INH-ODN activity related to base sequence derived from polymerization of INH-ODNs or their ability to complex with stimulatory CpG-oligonucleotides (ST-ODN). Furthermore, the 5' end of INH-ODNs may assume a particular loop configuration that may be needed for binding to a critical site on TLR9. Here, we show that 1) G-tetrads required for ODN stacking were compatible with INH-ODN activity but were not necessary; 2) there was no relationship between activity and self-association at endosomal pH; 3) there was no evidence for direct binding between ST-ODNs and INH-ODNs; 4) when a 3G sequence was disrupted, despite a preserved stem-loop formation, INH-ODN activity was abolished. These results support the conclusion that certain features of the primary linear sequence are critical for TLR9 inhibition, but changes in secondary structure or in ODN aggregation are irrelevant.

Sequence requirements for oligodeoxyribonucleotide inhibitory activity.

Inhibitory oligonucleotides (IN-ODN) differing from stimulatory CpG ODN (ST-ODN) by as few as two bases can block ST-ODN-induced proliferation, apoptosis protection and IL-6 secretion in B lymphocytes and the production of IL-12p40 by non-B cells. The main objective of this study was to determine the ODN sequence requirements for inhibition in mice. Starting with a strongly inhibitory 15-mer prototype phosphorothioate sequence, we tested the 60 sequences that differed from the prototype by one base, revealing the three areas that are critical for activity. Between these areas were the spacer sequences where base composition mattered little, but the number of bases was important. Truncation of three bases at the 3' end of the 15-mer and one at the 5' end was tolerated with minimal loss of activity. This approach yielded an 'optimal' sequence of 5' CC x notC notC xxGGGx or CC x notC notC xGGGxx 3', where x is any base. The sequence requirements for optimal inhibition of B cell responses to Type B (K) ODN and mixed splenocyte IL-12p40 responses to Type A (D) ODN were strikingly similar. Inhibition of ST-ODN by IN-ODN was competitive. A hypothetical model of the ODN-binding site is proposed. Synthetic IN-ODN with the sequence characteristics defined here should provide antidotes for excessive innate reactions to DNA.