Xenogeneic Human Tyrosinase DNA Vaccination Induces Specific Immune Response in Dogs with Advanced Malignant Melanoma

Introduction:  Xenogeneic melanosomal differentiation antigens, delivered in the form of a plasmid DNA vaccine, can overcome host immune ignorance/tolerance in preclinical animals to melanoma by: 1) generating humoral and cytotoxic T cell responses and 2) inducing protection from tumor challenge. Initial trials of human tyrosinase (huTyr) DNA vaccination of dogs with advanced malignant melanoma (Bergman et al , 2003) demonstrated safety and prolongation of survival with this therapeutic modality. We investigated antigen‐specific immunity in dogs receiving huTyr DNA vaccination.
Methods:  Three cohorts of three dogs each with advanced (WHO stage II‐IV) canine malignant melanoma (CMM) received four biweekly IM injections (dose levels 100, 500, or 1,500 ug, respectively/vaccination) of huTyr plasmid DNA via the Biojector2000 jet delivery device. Sera samples were taken before and after vaccination to detect specific antibody formation to huTyr by Enzyme‐Linked ImmunoSorbent Assays (ELISAs) and flow‐cytometry.
Results:  Three dogs have measurable, 2 to 4 fold huTyr‐specific antibody titers. Preliminary studies by flow‐cytometry have confirmed antibody response to huTyr by positive binding to endogenous human tyrosinase in SK‐Mel188 cells using post‐vaccinate serum.
Conclusions:  Xenogeneic (huTyr) DNA vaccination generates antigen‐specific humoral responses, which may partially explain the previously reported clinical efficacy and anti‐tumor responses. Ongoing studies include: 1) a comprehensive analysis by flow‐cytometry to detect huTyr‐specific antibodies and 2) a quantitative measure of potential cytotoxic T‐cell responses in these dogs by the DNA vaccination.     

Treatment of Metastatic Equine Melanoma with a Plasmid DNA Vaccine Encoding Streptococcus Pyogenes EMM55 Protein

A 19-year-old castrated male Arab/Quarter horse presented with an extensive history of cutaneous metastatic melanoma. Over a period of 8 months, a total of 8 doses of plasmid DNA vaccine expressing the Streptococcus pyogenes emm55 gene (pAc/emm55) were administered intratumorally at 300 μg/dose via a needless injector. Upon completion of the vaccination protocol, the size of the injected lesions, on average, were reduced by 40.3% from the initial size measurements. Lesions that were not injected were reduced by 47.6%. The overall reduction in total tumor burden was 42.3%. Tumor regression was also associated with the augmentation of antimelanoma IgG antibody response, thus implying that an induction of an effective antimelanoma response would be of great advantage in the management of equine melanoma.     

Inflammatory Response of Healthy Horses Subjected to Small Colon Enterotomy and Treated or Not With Heparin

The acute phase response is a response to injury and depends on the severity of the trauma. Heparin is routinely used for postsurgical treatment of horses to prevent abdominal adhesions; however, its effect on inflammation is unknown. This study aimed to assess systemic inflammatory response of horses subjected to small colon enterotomy and to evaluate heparin effects on postsurgical inflammation. Ten adult horses were subjected to small colon enterotomy and were assigned to a control or a treatment group. Both groups received prophylactic antibiotics and flunixin, and the treatment group received 150 IU/kg heparin subcutaneously after surgery and every 12 hours for five days. WBC counts, peritoneal fluid evaluation, determination of serum and peritoneal haptoglobin (Hp), and serum amyloid A (SAA) were performed before, 12 hours, and 1, 2, 4, 6, 10, and 14 days after enterotomy. Forty-eight hours after surgery, a significant increase in serum Hp was observed in the control group, and SAA concentrations increased significantly in the both groups between 24 hours, 48 hours, and 4 days after surgery. The SAA and serum Hp concentrations produced no significant differences between the groups. Peritoneal Hp increased significantly in the control group 4 days after surgery and was significantly higher in the control group than in the treated group 14 days after surgery. Serum Hp and SAA identified the acute phase response changes faster, however, were not able to identify differences between groups. Peritoneal Hp concentrations identified inflammatory differences between the groups 14 days after surgery; the difference suggests that heparin may act decreasing inflammation.     

Assessment of Gastric Ulceration and Gastrin Response in Horses with History of Crib-Biting

It was hypothesized that horses exhibiting crib-biting (CB) have a greater degree of gastric mucosal damage and higher serum gastrin response to concentrate feeding than non-crib-biting (NCB) horses. Eighteen mature horses, 9 CB and 9 NCB, were used to determine prevalence and severity of gastric mucosal damage and effect of concentrate feeding on circulating gastrin. Horses were maintained on pasture with free access to hay and fed a pelleted concentrate diet twice daily. Number of crib-bites and duration of cribbing bouts were recorded in a 24-hour period. Endoscopic examinations (EE) of the squamous mucosa were performed and gastric fluid sampled after 24 to 28 hour feed removal. Following EE, horses were returned to pasture for 72 hours. Blood was collected following 12-hour feed removal (0 minutes), and at 60 and 120 minutes after consuming 1 kg of concentrate. Mean number of crib bites in 24 hours was 1,558 ± 303 with CB peaking prior to and during the afternoon feeding (3:30 PM, P < .05). There were no differences in the number or severity of ulcers, prevalence of hyperkeratosis, or baseline gastric pH between CB and NCB. Serum gastrin concentration at 60 and 120 minutes was greater (P < .05) and tended to be greater (P < .06), respectively, in CB than in NCB horses following feeding of concentrate. Crib-biting behavior in horses maintained on pasture was not associated with gastric mucosal damage; however, consumption of concentrate feed resulted in greater serum gastrin concentration in CB horses.     

Usefulness of a Point-of-Care Analyzer to Measure Cardiac Troponin I and D-Dimer Concentrations in Critically Ill Horses With Gastrointestinal Diseases

Point-of-care (POC) systems for the joint measurement of Troponin and D-dimers have not been studied in horses. The aim of this study was to perform the validation of a POC system (AQT90 FLEX) for the measurement of cardiac troponin I (cTnI) and D-dimers in the serum of horses with gastrointestinal diseases. The main objective was to evaluate whether or not this system can distinguish healthy animals from diseased animals. A sample of 33 horses was included in the study: control group (n = 10) and horses with gastrointestinal disorders (n = 21), which were classified according to their outcome in survivors (subgroup A = 9) and nonsurvivors (subgroup B = 12). Considering the diagnosis of the process, ill horses were classified into three groups: inflammatory (I = 7), obstructive (O = 9), and strangulating diseases (S = 5). The clinical usefulness of AQT90 FLEX was validated by the study of linearity, coefficient of variation, and detection limits. Later, concentrations of D-dimers and cTnI were measured. A significant increase in both parameters was detected in ill animals (cTnI: control: 0.014 ± 0.01 μg/mL, survivors: 0.27 ± 0.37 μg/mL, nonsurvivors: 0.60 ± 1.21 μg/mL; D-dimers: control: 104.90 ± 30.82 ng/mL, survivors: 1,217.22 ± 1,213.28 ng/mL, nonsurvivors: 1,613.67 ± 1,426.75 ng/mL), although there were no statistically significant differences in concentrations according to diagnosis and outcome. In conclusion, AQT90 FLEX POC analyzer can be used in horses with gastrointestinal diseases to measure cTnI and D-dimer concentrations. It is a quick, practical, and minimally invasive tool that helps in determining the severity of illness.     

Use of Body-Mounted Inertial Sensors to Objectively Evaluate the Response to Perineural Analgesia of the Distal Limb and Intra-articular Analgesia of the Distal Interphalangeal Joint in Horses With Forelimb Lameness

Diagnostic analgesia of the distal interphalangeal (DIP) joint is theoretically helpful to localize the source of pain in the foot to the joint and/or navicular bursa. However, it has been suggested that potential diffusion of local anesthetic agent to nearby distal limb nerves may anesthetize other areas of the foot. The objective of this study was to compare the results of palmar digital (PD) and abaxial sesamoid (AS) nerve blocks to intra-articular anesthesia of the DIP joint in horses with distal forelimb lameness. Palmar digital nerve block (group 1) or PD and AS nerve blocks (group 2) were used to abolish digital pain in 22 horses. The following day lameness was again evaluated in all horses before and 2, 5, and 10 minutes after DIP joint anesthesia. All lameness evaluations were performed objectively with a body-mounted inertial sensor system (Lameness locator; Equinosis LLC, Columbia, MO). In group 1 horses, overall improvement in group lameness was the same after DIP joint block, but only six showed positive response after DIP joint analgesia, five after 2 minutes, and one after 5 minutes. In group 2 horses, overall improvement in lameness was less after DIP joint block, with seven showing a positive response after DIP joint analgesia, one after 2 minutes, four after 5 minutes, and two after 10 minutes. Intra-articular analgesia of the DIP joint and perineural analgesia of the digit result in overlapping but unequal areas of analgesia. In addition, a time-dependent response was observed after DIP joint block with full effect requiring 5–10 minutes.     

Use of a Grading System to Facilitate Treatment and Prognosis in Horses with Negative Palmar Angle Syndrome (Heel Collapse): 107 Cases

Negative palmar angle syndrome refers to the condition of progressive heel collapse and its consequences on gait and performance. Treatment and prognosis are facilitated by grading the severity of biomechanical disorder according to physical and radiographic features. Although negative palmar angle syndrome in grades I (mild) and II (moderate) can be corrected with trimming and routine shoeing, grades III (severe) and IV (complicated by flexor contracture) require more intensive mechanical intervention and patience—however, comfort and function can be improved immediately.     

Effect of Immunization with Plasmid DNA Encoding for Rinderpest Virus Matrix Protein on Systemic Rinderpest Virus Infection in Rabbits

Plasmid vaccine pBK-CMVMP1LC113 expressing the matrix (M) gene of rinderpest virus was assessed for its potential to protect rabbits against a lethal viral challenge. Rabbits immunized with plasmids expressing the M gene were not protected when challenged with lapinized rinderpest virus, despite the production of anti-M antibodies, while rabbits immunized with rinderpest tissue culture vaccine were completely protected from a lethal challenge with lapinized rinderpest virus. The plasmid vaccine also had no significant effect on the lymphopenia in challenged rabbits. The results indicate that rinderpest M protein does not have a protective role in rinderpest infection.     

Development of Immune Response to Experimental Bovine Trichophyton vervucosum Infection

Abstract— Cell mediated and humoral immune responses to experimental Trichophyton verrucosum infection were assessed by sequential cutaneous biopsies, antibody assessments and microscopic monitoring of fungal presence. Histopathologic examination showed the accrual of lymphocytes and other inflammatory cells in the dermis of infected sites. Immunoperoxidase staining of frozen sections with monoclonal antibody preparations revealed an influx of macrophages, BoCD4+ and B0CD8+ lymphocytes and γδ T cells from the 5th day to the 33rd day of infection. A moderate influx of B cells was observed. Protein G-colloidal gold staining revealed the presence of immunoglobulins in the dermis and superficial epidermal layers. Trichophyton specific serum antibodies appeared between days 33 and 55. Microscopic assessment of infected tissues revealed an increase in T, verrucosum elements (mycelium and ectothrix spores) from days 19 to 55. Fungal elements in infected areas did not decrease until after both humoral and cell mediated elements of the immune response were established. These responses imply a combination of cell mediated and humoral events were associated with T. verrucosum immunity and clearance in the calf. Résumé— Le réponse immunitaire humorale et cellulaire a une infection expérimentale àT. verrucosum a été appréciée par des biospsies cutanées successives, des dosages d'anticorps et la recherche microscopique de champignons. L'examen histopathologique a montré un afflux de lymphocytes et d'autres cellules inflammatoires dane le derme des sites infectés. Les marquages en immunopéroxydase par un anticorps monoclonal de coupes congelées a montré un influx de macrophages, lymphocytes BoCD4+ et BoCD8+ et des cellules T γδ, du 5e au 33e jour de l'infection. Un marquage par une protéine G—or colloidal a révélé d'immunogolglobulines dans le derme et les couches supéerficielles de l'épiderme. Les anticorps spécifiques de Trichophyton sont apparus entre 33 et 55 jours. L'examen microscopique des tissus infectés a révélé une augmentation du nombre d'éléments de T. verrucosum (mycelium et spores ectothrix) du 19e au 55e jours. Les éléments fongiques dans les zones infectées n'ont pas diminué avant que les réponses humorales et cellulaires ne solent établies. Ces résponses impliquent qu'une coopération des réponses humorales et cellulaires étaient associées dans l'immunité et la défense contre T. verrucosum. Zusammenfassung— Die zellvermittelten und humoralen Immunantworten auf die experimentelle Infektion mit T. verrucosum wurden durch eine Serie von Hautbiopsien, Antikörperuntersuchungen und mikroskopischer Untersuchung auf das Vorhandensein von Pilzen ausgewertet. Die histopathologische Untersuchung zeigte eine Ansammlung von Lymphozyten und anderen Entzündungszellen in der Dermis der infizierten Stellen. Die Immunperoxidasefärbung der Gefrierschnitte mit monoklonalen Antikörperzubereitungen zeigte einen Influx von Makrophagen, BoCd4-und BoCD8-Lymphozyten und gamma-delta-T-Zellen vom 5. bis zum 33. Tag der Infektion. Es wurde auch ein mäßiger Influx von B-Zellen beobachtet. Die Protein-G-kolloidale Goldfärbung zeigte die Anwesenheit von Immunglobulinen in der Dermis und den oberflächlichen epidermalen Schichten. Trichophyton-spezifische Serumantikörper traten zwischen Tag 33 und 55 auf. Die mikroskopische Untersuchung infizierter Gewebe zeigte eine Zunahme von T. verrucosum-Bestandteilen (Myzel und exktothrixe Sporen) vom Tag 19 bis 55. Pilzteile in infizierten Bereichen verminderten sich weder, nachdem humorale, noch nachdem zellvermittelte Elemente der Immunantwort auftraten. Diese Reaktionen deuten an, daß eine Kombination von zellvermittelten und humoralen Vorgängen im Zusammenhang mit T. verrucosum-Immumtät und Abheilung beim Kalb vorliegt. Resumen Por medio de biposias cutáneas secuenciales, medida de anticuerpos y exámen microscópico de presencia de hongos, se estudió la respuesta inmunitaria de tipo humoral y celular producida por la infección experimental con T. verrucosum. El exámen histopatológico reveló la presencia de agregación de linfocitos y otras células inflamatorias procedentes de la dermis de los puntos infectados. La tintura por medio de inmunoperoxidasa de las secciones congeladas, con preparaciones monoclonales de anticuerpos, demostró un aflujo de macrófagos BoCD4 + y BoCD8 + linfocitos y linfocitos, Tαδ, desde el quinto hasta el día 33 la infección. También se observó un aflugo moderado de linfocitos B. La tintura aúrica de proteina coloidal G reveló la presencia de inmunoglobulinas en al dermis y capas superficiales de la epidermis. Los anticuerpos específicos para la especie Trichophyton aparecieron entre los días 33 y 55. El exámen microscópico de los tejidos afectados demostró un incremento, de los elementos füngicos T. verrucosum (micelio y esporas exótricas) desde los días 19 al 55. Los elementos fúngicos en áreas infectadas no disminuyeron hasta después del establecimiento de ambos tipos de respuesta inmunitaria, humoral y celular. Estas respuestas implican que la combinación de ciertos fenómenos de inmunidad celular y humoral, están relacionados con la desaparición y la inmunidad de la infección producia por T. verrucosum en el ternero.     

猪带绦虫TS11抗原基因接种小鼠的免疫应答

本实验以猪带绦虫 TS11抗原基因的真核表达型质粒 VTS11肌肉免疫注射 BAL B/ c小鼠 ,四甲基偶氮唑蓝 (MTT)比色法检测小鼠脾淋巴细胞刀豆蛋白 A(Con A)刺激的增殖反应及 IL- 2的诱生活性 ;用 EL ISA方法检测免疫小鼠 Ig G总量和特异性抗体水平 ,常规法检测外周血免疫细胞数量的动态变化。结果显示 VTS11免疫小鼠血清的特异性抗体滴度和 Ig G含量显著高于空白对照组小鼠 ;免疫小鼠脾脏淋巴细胞 Con A刺激增殖反应和 IL - 2诱生活性均比对照组小鼠显著增强 ;VTS11免疫小鼠的淋巴细胞和巨噬细胞等免疫细胞的数量也显著超过对照组。这表明 VTS11免疫小鼠 ,可诱导其产生特异性的细胞和体液免疫反应 ,VTS11具有很强的免疫激活作用 ,可望成为预防猪囊虫病的一种新型疫苗。     

Study of the local immune response to Fasciola hepatica in the liver and hepatic lymph nodes of goats immunised with a peptide of the Sm14 antigen

The nature of the local immune response was assessed studying the distribution of CD2⁺, CD4⁺, CD8⁺, γδ⁺ T lymphocytes, IgM⁺ B cells, IL-4⁺ and IFN-γ⁺ cells in the liver and hepatic lymph nodes (HLN) of goats immunised with a synthetic peptide of the Sm14 antigen from Schistosoma mansoni and challenged with Fasciola hepatica. A morphometric study of HLN was also carried out in order to evaluate the hyperplasia of lymphoid follicles. Despite the decrease in fluke burdens found in the immunised group (45.9%) respect to the infected control group, this difference was not statistically significant due to the high individual variability. In liver, a significant increase of CD2⁺, CD4⁺, CD8⁺, γδ⁺ T lymphocytes was found in the infected groups respect to the uninfected control and in the infected control respect to the immunised group. HLN showed a significant enlargement due to the hyperplasia of lymphoid follicles and infiltration of CD2⁺, CD4⁺, CD8⁺, γδ⁺ T lymphocytes in both infected groups respect to the uninfected control, with no significant differences between the infected control and immunised group. IFN-γ⁺ lymphoid cells was absent or very occasional in HLN where the number of IL-4⁺ cells was higher than that of IFN-γ, suggesting a polarized Th2 response in immunised and in infected control group.     

A Pilot Study to Assess the Feasibility of a Submaximal Exercise Test to Measure Individual Response to Cardiac Medication in Dogs with Acquired Heart Failure

Exercise testing is not commonly used in canine medicine because of several limitations. The aim of this study was to investigate the suitability of a treadmill test to measure the exercise capacity of untrained canine cardiac patients and to measure some biological parameters that might reflect the tolerance of dogs with heart failure to submaximal exercise. The exercise capacity of seven dogs with naturally occurring heart failure was evaluated before the institution of cardiac medication and 7 days after the beginning of the study. An additional re-examination was requested after 28 days. The exercise test was performed on a motorized treadmill at three different speeds (0.5 m/s, 1.0 m/s and 1.5 m/s). The following parameters were measured at the end of each stage and after 20 min recovery: heart rate, rectal temperature, glucose, lactate, aspartate aminotransferase, creatine kinase, Pvo ₂, Pvco ₂, pH, haematocrit, bicarbonate, sodium, potassium and chloride. Serum cardiac troponin-I was also measured at the beginning of the test and at the end of the recovery period. Owners’ perception reflected the ability of their dogs to exercise on the treadmill. Lactate level increased noticeably with the intensity of the exercise test, and its variation coincided with different exercise tolerance observed by the owners. Heart rate seemed to follow a similar trend in the few dogs presented in sinus rhythm. None of the remaining parameters appeared to be sensitive indicators of activity level in the dogs used in this study. The treadmill exercise test in dogs with acquired heart failure is feasible and might provide useful information for assessing individual response to cardiac medication. Lactate and heart rate seemed to reflect individual levels of exercise tolerance, although further studies are necessary to confirm the reliability and repeatability of this test.     

Development of a Latex Agglutination Test Using The Major Epitope Domain of Glycoprotein E of Pseudorabies Virus Expressed in <Emphasis Type="Italic">E. coli</Emphasis> to Differentiate Between Immune Responses in Pigs Naturally Infected or Vaccinated with Pseudorabies Virus

A 0.8 kb DNA fragment encoding the major epitope domain of glycoprotein E (gE) of pseudorabies virus (PRV) was inserted downstream of the T7 promoter of an expression vector, pET-28b, to yield the recombinant plasmid pETgE804. After induction by isopropy1-β-D-thiogalactopyranoside (IPTG), a high level expression of fusion protein was obtained. SDS-PAGE and western immunoblotting analysis showed that the fusion protein was 38 kDa and could bind with antisera against PRV. The protein existed mainly in the form of the inclusion body. After being denatured and renatured, the protein was used to prepare the latex antigen. The concentration of antigen, temperature and time for sensitization were optimized. The latex agglutination test (LAT) was able to differentiate sera of PRV-infected pigs from those of gE-deletion vaccine-immunized pigs. The diagnostic specificity and sensitivity of the developed gE latex agglutination test (gE-LAT) were also evaluated by using sets of sera. The diagnostic specificity and diagnostic sensitivity of the gE-LAT were 96.77% and 95.76%, respectively. For comparison between gE-LAT and a commercial blocking enzyme-linked immunosorbent assays (ELISA), 260 serum samples were tested. The coincidence frequency of both assays was 96.94% (252/260). No significant difference was found between the two methods (p>0.05). For comparison between the abilities of gE-LAT and gE-ELISA to detect sera with low titres of gE-specific antibody, 66 sera from 22 pigs were tested. The data indicate that the gE-LAT is of similar sensitivity to gE-ELISA. These results indicate that gE-LAT using recombinant gE might be very useful as a routine screening method for the differential diagnosis of PRV infection.