Post-Vaccination Delivery of CpG ODNs Enhances the Th2-Associated Protective Immunity of the Smallpox DNA Vaccine.

Potential threat of smallpox bioterrorism and concerns related to the adverse effects of currently licensed live-virus vaccines suggest the need to develop novel vaccines with better efficacy against smallpox. Use of DNA vaccines containing specific antigen-encoding plasmids prevents the risks associated with live-virus vaccines, offering a promising alternative to conventional smallpox vaccines. In this study, we investigated the efficiency of toll-like receptor (TLR) ligands in enhancing the immunogenicity of smallpox DNA vaccines. BALB/c mice were immunized with a DNA vaccine encoding the vaccinia virus L1R protein, along with the cytosine-phosphate-guanine (CpG) motif as a vaccine adjuvant, and their immune response was analyzed. Administration of B-type CpG oligodeoxynucleotides (ODNs) as TLR9 ligands 24 h after DNA vaccination enhanced the Th2-biased L1R-specific antibody immunity in mice. Moreover, B-type CpG ODNs improved the protective effects of the DNA vaccine against the lethal Orthopoxvirus challenge. Therefore, use of L1R DNA vaccines with CpG ODNs as adjuvants is a promising approach to achieve effective immunogenicity against smallpox infection.

Pretreatment of rhesus monkeys with transdermal patches containing physostigmine and procyclidine: implications of the delivery system for the potential application against VX nerve agent intoxication in humans.

Physostigmine (Phs) is a reversible inhibitor of acetylcholinesterase (AChE) that penetrates the blood-brain barrier (BBB) and could be used to protect the central nervous system (CNS) against the effects of nerve agents. For prophylactic effectiveness, long, steady, and adequate inhibition of AChE activity by Phs is needed to broadly protect against the CNS effects of nerve agents. Here, we evaluated the efficacy of transdermal patches containing Phs and procyclidine (PC) as prophylactic agents. Patches (25 cm2) containing 4.4 mg Phs and 17.8 mg PC had a protective ratio of approximately 78.6-fold in rhesus monkeys challenged with VX nerve agent and given an antidote. Physiologically based pharmacokinetic model in conjunction with an indirect pharmacodynamic (PBPK/PD) was developed for Phs and scaled to rhesus monkeys. The model was able to reproduce the concentration profile and inhibitory effect on AChE of Phs in monkeys, as evidenced by correlation coefficients of 0.994 and 0.992 for 25 cm2 and 49 cm2 patches, respectively (i.e., kinetic data), and 0.989 and 0.968 for 25 cm2 and 49 cm2 patches, respectively (i.e., dynamic data). By extending the monkey PBPK/ PD model to humans, the effective human dose was predicted to be five applications of a 25 cm2 patch (i.e., 22 mg Phs), and two applications of a 49 cm2 patch (i.e., 17.4 mg Phs). Therefore, given that patch application of Phs in rhesus monkeys has a prolonged effect (namely, AChE inhibition of 19.6% for the 25 cm2 patch and 23.0% for the 49 cm2 patch) for up to 216 h, patch formulation of Phs may provide similar protection against nerve agent intoxication in humans.

A multipathogen DNA vaccine elicits protective immune responses against two class A bioterrorism agents, anthrax and botulism.

The potential use of biological agents has become a major public health concern worldwide. According to the CDC classification, Bacillus anthracis and Clostridium botulinum, the bacterial pathogens that cause anthrax and botulism, respectively, are considered to be the most dangerous potential biological agents. Currently, there is no licensed vaccine that is well suited for mass immunization in the event of an anthrax or botulism epidemic. In the present study, we developed a dual-expression system-based multipathogen DNA vaccine that encodes the PA-D4 gene of B. anthracis and the HCt gene of C. botulinum. When the multipathogen DNA vaccine was administered to mice and guinea pigs, high level antibody responses were elicited against both PA-D4 and HCt. Analysis of the serum IgG subtype implied a combined Th1/Th2 response to both antigens, but one that was Th2 skewed. In addition, immunization with the multipathogen DNA vaccine induced effective neutralizing antibody activity against both PA-D4 and HCt. Finally, the protection efficiency of the multipathogen DNA vaccine was determined by sequential challenge with 10 LD50 of B. anthracis spores and 10 LD50 of botulinum toxin, or vice versa, and the multipathogen DNA vaccine provided higher than 50% protection against lethal challenge with both high-risk biothreat agents. Our studies suggest the strategy used for this anthrax-botulinum multipathogen DNA vaccine as a prospective approach for developing emergency vaccines that can be immediately distributed on a massive scale in response to a biothreat emergency or infectious disease outbreak. Key points • A novel multipathogen DNA vaccine was constructed against anthrax and botulism. • Robust immune responses were induced following vaccination. • Suggests a potential vaccine development strategy against biothreat agents.

A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein.

Vaccines and therapeutics are urgently needed for the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we screen human monoclonal antibodies (mAb) targeting the receptor binding domain (RBD) of the viral spike protein via antibody library constructed from peripheral blood mononuclear cells of a convalescent patient. The CT-P59 mAb potently neutralizes SARS-CoV-2 isolates including the D614G variant without antibody-dependent enhancement effect. Complex crystal structure of CT-P59 Fab/RBD shows that CT-P59 blocks interaction regions of RBD for angiotensin converting enzyme 2 (ACE2) receptor with an orientation that is notably different from previously reported RBD-targeting mAbs. Furthermore, therapeutic effects of CT-P59 are evaluated in three animal models (ferret, hamster, and rhesus monkey), demonstrating a substantial reduction in viral titer along with alleviation of clinical symptoms. Therefore, CT-P59 may be a promising therapeutic candidate for COVID-19.

Immunogenicity and Biodistribution of Anthrax DNA Vaccine Delivered by Intradermal Electroporation.

PURPOSE: Anthrax is a lethal bacterial disease caused by gram-positive bacterium Bacillus anthracis and vaccination is a desirable method to prevent anthrax infections. In the present study, DNA vaccine encoding a protective antigen of Bacillus anthracis was prepared and we investigated the influence of DNA electrotransfer in the skin on the induced immune response and biodistribution. METHODS AND RESULTS: The tdTomato reporter gene for the whole animal in vivo imaging was used to assess gene transfer efficiency into the skin as a function of electrical parameters. Compared to that with 25 V, the transgene expression of red fluorescent protein increased significantly when a voltage of 90 V was used. Delivery of DNA vaccines expressing Bacillus anthracis protective antigen domain 4 (PAD4) with an applied voltage of 90 V induced robust PA-D4-specific antibody responses. In addition, the in vivo fate of anthrax DNA vaccine was studied after intradermal administration into the mouse. DNA plasmids remained at the skin injection site for an appropriate period of time after immunization. Intradermal administration of DNA vaccine resulted in detection in various organs (viz., lung, heart, kidney, spleen, brain, and liver), although the levels were significantly reduced. CONCLUSION: Our results offer important insights into how anthrax DNA vaccine delivery by intradermal electroporation affects the immune response and biodistribution of DNA vaccine. Therefore, it may provide valuable information for the development of effective DNA vaccines against anthrax infection.

Development of an Equine Antitoxin by Immunizing the Halla Horse with the Receptor-Binding Domain of Botulinum Neurotoxin Type A1.

Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are the most toxic substances known. However, the number of currently approved medical countermeasures for these toxins is very limited. Therefore, studies on therapeutic antitoxins are essential to prepare for toxin-related emergencies. Currently, more than 10,000 Halla horses, a crossbreed between the native Jeju and Thoroughbred horses, are being raised in Jeju Island of Korea. They can be used for equine antitoxin experiments and production of hyperimmune serum against BoNT/A1. Instead of the inactivated BoNT/A1 toxoid, Halla horse was immunized with the receptor-binding domain present in the C-terminus of heavy chain of BoNT/A1 (BoNT/A1-HCR) expressed in Escherichia coli. The anti-BoNT/A1-HCR antibody titer increased rapidly by week 4, and this level was maintained for several weeks after boosting immunization. Notably, 20 µL of the week 24 BoNT/A1-HCR(-immunized) equine serum showed an in vitro neutralizing activity of over 8 international unit (IU) of a reference equine antitoxin. Furthermore, 20 µL of equine serum and 100 µg of purified equine F(ab')2 showed 100% neutralization of 10,000 LD50 in vivo. The results of this study shall contribute towards optimizing antitoxin production for BoNT/A1, which is essential for emergency preparedness and response.

Intradermal immunization with botulinum neurotoxin serotype E DNA vaccine induces humoral and cellular immunity and protects against lethal toxin challenge.

Botulinum neurotoxins (BoNTs) produced by the spore-forming, gram-positive, anaerobic bacterium Clostridium botulinum are the most toxic substances known and cause botulism, flaccid paralysis, or death. Owing to their high lethality, BoNTs are classified as category A agents by the Centers for Disease Control (CDC). Currently, there are no vaccines available to protect against BoNTs, so the rapid development of a safe and effective vaccine is important. DNA-based vaccines have recently drawn great attention because they can be developed quickly and can be applied in mass vaccination strategies to prevent disease outbreaks. Here, we report on the immunogenic and protective efficacy of a DNA vaccine, encoding a 50-kDa carboxy-terminal fragment of the BoNT serotype E heavy chain, which is delivered via an intradermal route. This plasmid DNA vaccine induced robust humoral and cellular BoNT/E-specific immune responses and completely protected animals against lethal challenge with BoNT/E. These results not only indicate that DNA vaccines could be further developed as safe and effective candidates for vaccines against BoNTs but also suggest a possible approach for developing vaccines that protect against bio-threat toxins.

A mutated recombinant subunit vaccine protects mice and guinea pigs against botulinum type A intoxication.

Botulinum neurotoxins (BoNTs) are the most potent toxins to mammals. A toxoid vaccine was previously used for prevention of botulinum intoxication; however, this vaccine is no longer available. Currently, no approved botulinum vaccines are available from the Food and Drug Administration (FDA). Recently, a recombinant host cell receptor-binding subunit created for use as a potential vaccine completed phase 2 clinical trials. The current study designed a vaccine candidate against BoNT type A (BoNT/A) using a structural design. Our vaccine candidate was the BoNT/A heavy chain C-terminal region (HCR) that contained the point mutation BA15 (R1269A) within the ganglioside-binding site. A Biacore affinity test showed that the affinity of BA15 for ganglioside GT1b was 100 times lower than that of the HCR. A SNAP25 cleavage assay revealed that immunized sera blocked SNAP25 cleavage of the BoNT/A toxin via BA15. In an in vivo experiment, mice and guinea pigs immunized with BA15 produced neutralizing antibodies that protected against 3,000 LD50 of BoNT/A. In conclusion, the results of both in vitro and in vivo assays showed that our BA15 vaccine candidate was similar to the recombinant host cell receptor-binding subunit vaccine. The inability of BA15to bind ganglioside shows that BA15 is a potential safe vaccine candidate.

A novel staphylococcal enterotoxin B subunit vaccine candidate elicits protective immune response in a mouse model.

Staphylococcal enterotoxin B (SEB), produced by the gram-positive bacterium Staphylococcus aureus, is responsible for food poisoning and toxic shock syndrome, and is considered a potential bioterrorism agent. Unfortunately, still now no approved vaccines are available against SEB. In this study, we constructed a series of nontoxic SEB mutants (mSEBs) and examined whether these mSEBs provide protective immunity against SEB challenge. These mSEB vaccine candidates did not demonstrate superantigen activity in mouse splenocyte cultures. Immunization with the vaccine candidates triggered the production of IgG-antibodies with neutralizing activity. In addition, increased production of IgG1 and IgG3 was observed after immunization, which signifies both Th1- and Th2-induced immune responses. Among the vaccine candidates tested, S9, a double mutant (N23A and Y90A) and S19, a quadruple mutant (N23A, Y90A, R110A, and F177A), demonstrated complete protection against a lethal SEB challenge. Altogether, our results strongly suggest that these mSEBs could be an effective recombinant SEB vaccine candidates for further/future preclinical and clinical studies.

Increased expression of vascular endothelial growth factor in neo-vascularized canine brain tissue.

This retrospective study was done to characterize the levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1 (HIF-1α) in dog brains with neo-vascularization in the cerebral cortex of frontal, temporal, and parietal lobe by using immunohistochemistry (IHC) and Western blot. In neo-vascularized (NV) brains, we analyzed the number and area of blood vessels and the expression of VEGF and HIF-1α. The IHC results showed that the number and area of blood vessels, as assessed by immunolabeling for von Willebrand factor, was higher in the NV brain than in the control brain. The Western blot results showed that the level of VEGF was increased, predominantly in NV brain of the cerebral cortex relative to the clinically normal cerebral cortex, whereas the expression of HIF-1α in NV brains was not different from the control brains. Our study showed that dilatation of vessels and development of new vessels in the cerebral cortex were observed in cases of canine CNS disease and found increased expression of VEGF in canine brains with neo-vascularization.

An effective strategy for increasing the radiosensitivity of Human lung Cancer cells by blocking Nrf2-dependent antioxidant responses.

Radiotherapy and chemotherapeutic agents can effectively induce apoptosis through generation of reactive oxygen species (ROS). Cancer cells frequently express high levels of ROS-scavenging enzymes, which confer resistance to ROS-mediated cell death. Keap1 (Kelch-like ECH-associated protein 1) sequesters and promotes the degradation of the antioxidant response element-binding transcription factor Nrf2 (nuclear factor erythroid-2-related factor 2). In non-small-cell lung cancer (NSCLC) cell lines and NSCLC patients, Keap1 is often present as a biallelic mutant that results in constitutive activation of Nrf2 function, which contributes to cytoprotection against oxidative stress and xenobiotics. To identify small molecules that inhibit antioxidant responses and increase apoptotic death after radiotherapy, we screened a chemical library containing 8000 synthetic compounds using a cell-based luciferase assay system. 4-(2-Cyclohexylethoxy)aniline (IM3829) inhibited the increase in Nrf2-binding activity and expression of the Nrf2 target genes induced by treatment with tertiary butylhydroquinone or radiation. Combined treatment with IM3829 and radiation significantly inhibited clonogenic survival of H1299, A549, and H460 lung cancer cells. IM3829 significantly increased ROS accumulation in irradiated cells compared with cells exposed to radiation alone and led to apoptotic cell death, as confirmed by caspase-3 and PARP cleavage. In mice bearing H1299 or A549 lung cancer xenografts, IM3829 together with radiation inhibited tumor growth more effectively than radiation alone. Our findings suggest that IM3829 could be a promising radiosensitizer in lung cancer patients, particularly those with high expression of Nrf2.

Acriflavine enhances radiosensitivity of colon cancer cells through endoplasmic reticulum stress-mediated apoptosis.

Radiotherapy (RT) is one of the most effective tools in the clinical treatment of cancer. Because the tumor suppressor p53 plays a central role in radiation-mediated responses, including cell cycle-arrest and apoptosis, a number of studies have suggested that p53 could be a useful therapeutic target of anti-cancer agents. Accordingly, we sought to discover a new agent capable of increasing p53 activity. HCT116 colon cancer cells, containing wild-type p53, were stably transfected with a p53 responsive-luciferase (p53-Luc) reporter gene. A cell-based high-throughput screen of 7920 synthetic small molecules was performed in duplicate. Of the screened compounds, acriflavine (ACF) significantly increased p53-Luc activity in a concentration-dependent manner without causing toxicity. Pretreatment with ACF enhanced the induction of p53 protein expression and phosphorylation on serine 15 by γ-irradiation. Clonogenic assays showed that ACF pretreatment also potentiated radiation-induced cell death. The combination of irradiation and ACF treatment induced mitochondrial release of cytochrome c and significant activation of caspase-3 with PARP cleavage in colon cancer cells, demonstrating typical apoptotic cell death. Combined treatment with ACF and radiation increased the expression of Bax and Bad, while decreasing expression of Bcl-2. In addition, the ACF/radiation treatment combination induced endoplasmic reticulum (ER) stress responses mediated by IRE1α (inositol-requiring transmembrane kinase and endonuclease 1α), eIF-2α (eukaryotic initiation factor 2α), caspase-2/12, and CHOP (C/EBP homologous protein). The knockdown of IRE1α by siRNA inhibited the apoptotic cell death induced by ACF/radiation treatment. In vivo studies showed that combined treatment with ACF and radiation significantly inhibited the growth of tumors in colorectal cancer xenografted mice. These results indicate that ACF acts through p53-dependent mitochondrial pathways and ER stress signals, and could be a promising radiosensitizer.

Pro- and anti-inflammatory cytokine expression and histopathological characteristics in canine brain with traumatic brain injury.

We analyzed the expression level and cellular localization of pro- and anti-inflammatory cytokines and histopathologically characterized canine traumatic brain injury (TBI). Canine TBI brains revealed subarachnoid and cerebral cortical hemorrhage, neutrophilic infiltration, neuronal necrosis, astrocytosis, and vasogenic edema. Immunohistochemical evaluations suggested that both pro-inflammatory cytokines [interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α] and anti-inflammatory cytokines [IL-10 and transforming growth factor-beta (TGF-ß)] were highly expressed in neurons and neutrophils. In particular, the highest magnitude of expression was identified for IL-1ß and TGF-ß. This data helps describe the pathologic characteristics of canine TBI, and may help in the design of potential therapeutic approaches to control secondary damage by inflammatory cytokines.

Serial expression of hypoxia inducible factor-1α and neuronal apoptosis in hippocampus of rats with chronic ischemic brain.

OBJECTIVE: The purpose of this study is to investigate serial changes of hypoxia-inducible factor 1α (HIF-1α), as a key regulator of hypoxic ischemia, and apoptosis of hippocampus induced by bilateral carotid arteries occlusion (BCAO) in rats. METHODS: Adult male Wistar rats were subjected to the permanent BCAO. The time points studied were 1, 2, 4, 8, and 12 weeks after occlusions, with n=6 animals subjected to BCAO, and n=2 to sham operation at each time point, and brains were fixed by intracardiac perfusion fixation with 4% neutral-buffered praraformaldehyde for brain section preparation. Immunohistochemistry (IHC), western blot and terminal uridine deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were performed to evaluate HIF-1α expression and apoptosis. RESULTS: In IHC and western blot, HIF-1α levels were found to reach the peak at the 2nd week in the hippocampus, while apoptotic neurons, in TUNEL assay, were maximal at the 4th week in the hippocampus, especially in the cornu ammonis 1 (CA1) region. HIF-1α levels and apoptosis were found to fluctuate during the time course. CONCLUSION: This study showed that BCAO induces acute ischemic responses for about 4 weeks then chronic ischemia in the hippocampus. These in vivo data are the first to show the temporal sequence of apoptosis and HIF-1α expression.

Histopathological retrospective study of canine renal disease in Korea, 2003~2008.

Renal disease includes conditions affecting the glomeruli, tubules, interstitium, pelvis, and vasculature. Diseases of the kidney include glomerular diseases, diseases of the tubules and interstitium, diseases of renal pelvis, and developmental abnormalities. Renal tissue samples (n = 70) submitted to the Department of Veterinary Pathology of Konkuk University from 2003 to 2008 were included in this study. Tissue histopathology was performed using light microscopy with hematoxylin and eosin stains. Masson's trichrome, Congo Red, and Warthin starry silver staining were applied in several individual cases. Glomerular diseases (22.9%), tubulointerstitial diseases (8.6%), neoplastic diseases (8.6%), conditions secondary to urinary obstruction (24.3%), and other diseases (35.7%) were identified. Glomerulonephritis (GN) cases were classified as acute proliferative GN (5.7%), membranous GN (4.3%), membranoproliferative GN (4.3%), focal segmental GN (2.9%), and other GN (4.2%). The proportion of canine GN cases presently identified was not as high as the proportions identified in human studies. Conversely, urinary obstruction and end-stage renal disease cases were relatively higher in dogs than in human populations.

Distribution and characterization of IL-10-secreting cells in lymphoid tissues of PCV2-infected pigs.

Distribution and characterization of interlukin-10 (IL-10)-secreting cells in lymphoid tissues of pigs naturally infected with porcine circovirus type 2 (PCV2) were evaluated in accordance with PCV2 antigen detection. After screening a total of 56 pigs showing the symptoms of postweaning multisystemic wasting syndrome (PMWS), 15 pigs were PCV2 positive and 5 pigs, which showed stronger positive signals over multiples tissues were further investigated. This study showed that in PCV2-infected lymphoid tissues, particularly mandibular lymph node, spleen and tonsil, IL-10 expression was mainly localized in T-cell rich areas but rarely in B cell rich areas. IL-10 was highly expressed in bystander cells but rarely in PCV2-infected cells. Elevated IL-10 expression was predominantly associated with T cells, but rarely with B cells or with macrophages. The results of this study provide evidence for the role of IL-10 in chronic PCV2 infection and its relation to PCV2 antigen in affected tissues. Constantly elevated levels of IL-10 lead to immunosuppression in persistent and chronic viral infections. The increased IL-10 expression observed in PCV2 infection in this study suggests that IL-10-mediated immunosuppression may play an important role in the pathogenesis and maintenance of naturally occurring PCV2 infection.

Canine classical seminoma: a specific malignant type with human classifications is highly correlated with tumor angiogenesis.

BACKGROUND: Human seminoma is classified as classical seminoma (SE) and spermatocytic seminoma (SS). Human SE is known to be more malignant and metastasizing more frequently than SS. Tumor angiogenesis is highly related with tumor progression and metastasis, with microvessel density (MVD) being an important parameter of metastatic potential. Canine seminoma is not yet well-established as SE or SS type including correlation with angiogenesis. We classified canine SE and SS, and then compared them to tumor associated vessels. METHODS: Twenty-three cases of canine seminomas (2 intratubular, 9 diffuse, and 12 intratubular/diffuse seminomas showing both intratubular and diffuse patterns) were classified as SE or SS by immunohistochemistry (IHC) using monoclonal antibody against PLAP and by PAS stain. The histopathological data were then compared to see if there was a correlation with SE or SS. Angiogenesis of seminomas were evaluated by immunohistochemical assay using polyclonal antibody against Von Willebrand factor (vWF) and by calculating the means of MVD, vessels area and perimeters using computerized image analysis. Statistical Package for Social Sciences (SPSS) program was used for various statistical analyses. RESULTS: The numbers of PLAP+/PAS+ canine SEs were 8/23 (34.8%) and PLAP-/PAS- SSs were 15/23 (61.2%). All SE cases (8/8, 100%) were intratubular/diffuse types. SS types included 2 intratubular (2/15, 13.3%), 9 diffuse (9/15, 60%), and 4 intratubular/diffuse (4/15, 26.7%) types. MVD and vascular parameters in SEs were significantly higher than in SSs, showing the highest value in the intratubular/diffuse type. Seminomas observed with neoplastic cells invasion of vessels presented higher perimeter and area values than seminomas without conformed neoplastic cells invasion. CONCLUSION: In this study, we demonstrated a positive relationship between canine SE and tumor angiogenesis. Furthermore, we also showed that a tumor cells invasion of vessels were a correlated vascular parameter. Although metastasis of canine seminomas has rarely been reported, our results support that canine SE could have high metastatic potential similar to the human counterpart. Further studies are required to clarify the relationship between canine SE and clinical data with metastatic factors.

Characterization of canine oral papillomavirus by histopathological and genetic analysis in Korea.

In August 2008, forty dogs out of 400 developed oral warts in a breeding farm in Korea. Canine oral papilloma infection is a common disease in dogs. However, there has been no report of an outbreak of canine oral papillomavirus (COPV) in a group of dogs or in dog breeding farms in Korea, and the genetic analysis of COPV in Korea has yet to be performed. This study diagnosed canine oral papilloma from the oral samples of these dogs based on histopathological examination and immunohistochemistry. Polymerase chain reaction was applied to amplify the corresponding products using preexisting primer sets for COPV and a universal human papillomavirus targeting L1 gene. Further genetic analysis of the major viral capsid gene L1 confirms the sequences of Korean COPV, which shows a close relationship to previously reported COPV. This study describes the histopathological and immunohistochemical characteristics of canine oral papilloma in a group of breeding dogs in Korea and discloses the complete L1 gene sequences of Korean COPV.

Comparative immunohistochemical characterization of canine seminomas and Sertoli cell tumors.

Primary testicular tumors are the most common causes of cancer in male dogs. Overall, the majority of canine patients should be cured by testicular surgery. However, tumor markers are not well-known in veterinary medicine. We sought to determine using immunohistochemistry whether the combined human testicular tumor markers (placental alkaline phosphatase, OCT3/4, CD30, alpha-fetoprotein, inhibin-alpha, vimentin, c-KIT, and desmin) are expressed in canine seminomas and Sertoli cell tumors (SCTs). We examined 35 canine testicular tumors, 20 seminomas and 15 SCTs. c-KIT was expressed markedly in canine seminomas. Both inhibin-alpha and vimentin were expressed significantly in canine SCTs. The results of this study demonstrate differences and similarities between tumor marker expression of testicular tumors in dogs and humans. All the main markers in current routine use are discussed as well as potential useful markers for benign and malignant tumors, and tumor progression.

Effects of T lymphocytes, interleukin-1, and interleukin-6 on renal fibrosis in canine end-stage renal disease.

Canine end-stage renal disease (ESRD) is defined as the almost complete failure of renal function or irreversible destruction and is characterized by extensive glomerular sclerosis, tubular atrophy, interstitial inflammation, and fibrosis. Renal fibrosis is a common pathway leading to kidney failure. Infiltrating immunocytes in the end-stage kidney and several related factors are involved in renal fibrogenesis. A total of 18 renal tissue samples were obtained from canine patients with ESRD using biopsy and necropsy procedures. The extent of renal fibrosis was histopathologically examined by Masson trichrome staining. T-cell and B-cell localization and macrophage lineages were determined by immunohistochemical staining. Additionally, interleukin-1 (IL-1), IL-2, and IL-6 levels in the canine ESRD kidney were immunohistochemically evaluated and compared with expression patterns in the normal kidney. Significant fibrosis and infiltrating immunocytes consistent with lymphocytes were observed. Although the B-cell count was increased in the end-stage kidney, immunostaining patterns disclosed a marked increase in the number of CD3(+) cells. Furthermore, the remarkable increase in IL-1 and IL-6 levels suggests that T cells in the kidneys of dogs with ESRD spontaneously express these cytokines. In this study, the correlation between the degree of renal fibrosis and cytokines in canine ESRD was examined. The present study shows that T lymphocytes and IL-6 play important roles in renal fibrosis.