Revisiting the use of sPLA2-sensitive liposomes in cancer therapy.

The first developed secretory phospholipase A2 (sPLA2) sensitive liposomal cisplatin formulation (LiPlaCis®) is currently undergoing clinical evaluation. In the present study we revisit and evaluate critical preclinical parameters important for the therapeutic potential and safety of platinum drugs, here oxaliplatin (L-OHP), formulated in sPLA2 sensitive liposomes. We show the mole percentage of negatively charged phospholipid needed to obtain enzyme-sensitivity for saturated systems is ≥25% for 16-carbon chain lipid membranes, and >40% for 18-chain lipid membranes, which was surprising as 25% is used clinically in LiPlaCis®. Efficient sPLA2-dependent growth inhibition of colorectal cancer cells was demonstrated in vitro, where cell membrane degradation and cytolysis depends on the sensitivity of the formulation towards the enzyme and is governed by the amount of lysolipids generated and the presence of serum proteins. We found that serum proteins did not affect the lipase activity of the enzyme towards the membranes but instead sequester the lysolipid byproducts consequently inhibiting their detergent-like cytotoxic properties. In vivo therapeutic potential and safety of the liposomes was investigated in nude mice bearing sPLA2-deficient FaDu squamous carcinoma and sPLA2-expressing Colo205 colorectal adenocarcinoma. After intravenous injections, the tumor growth was suppressed for liposomal L-OHP relative to free drug, but only a weak response was observed for both slow- and fast-releasing sPLA2-sensitive formulations compared to non-sensitive liposomes. Also, the mice did not show longer survival. In turn, for the highly sPLA2-sensitive liposomes, multiple high doses caused petechial cutaneous hemorrhages, along with multifocal hepatonecrotic lesions, suggestive of premature activation in skin and liver irrespective of sPLA2-status of the tumor engraft. These results indicate that although liposomal carriers can improve the antitumor efficacy of platinum drugs, sPLA2-triggered release suffers from a narrow therapeutic index and has safety concerns.

Modelling severe Staphylococcus aureus sepsis in conscious pigs: are implications for animal welfare justified?

BACKGROUND: A porcine model of haematogenous Staphylococcus aureus sepsis has previously been established in our research group. In these studies, pigs developed severe sepsis including liver dysfunction during a 48 h study period. As pigs were awake during the study, animal welfare was challenged by the severity of induced disease, which in some cases necessitated humane euthanasia. A pilot study was therefore performed in order to establish the sufficient inoculum concentration and application protocol needed to produce signs of liver dysfunction within limits of our pre-defined humane endpoints. METHODS: Four pigs received 1 × 10(8) cfu/kg BW of S. aureus, and two controls were sham inoculated with saline. A fixed infusion rate of 3 mL/min was used, while the inoculum concentration, i.e., the dose volume, was changed between the pigs. The following dose volumes were used: 10 mL (n = 1), 20 mL (n = 2), and 30 mL (n = 1), corresponding to infusion durations of 3.33, 6.66, and 10 min at dose rates of 3 × 10(7), 1.5 × 10(7), and 1 × 10(7) cfu/min/kg BW, respectively. Blood samples were drawn for complete blood count, clinical chemistry, and inflammatory markers before and every 6 h after inoculation. Prior to euthanasia, a galactose elimination capacity test was performed to assess liver function. Pigs were euthanised 48 h post inoculation for necropsy and histopathological evaluation. RESULTS: While infusion times of 6.66 min, and higher, did not induce liver dysfunction (n = 3), the infusion time of 3.33 min (n = 1) caused alterations in parameters similar to what had been seen in our previous studies, i.e., increasing bilirubin and aspartate aminotransferase, as well as histopathological occurrence of intravascular fibrin split products in the liver. This pig was however euthanised after 30 h, according to humane endpoints. CONCLUSIONS: A usable balance between scientific purpose and animal welfare could not be achieved, and we therefore find it hard to justify further use of this conscious porcine sepsis model. In order to make a model of translational relevance for human sepsis, we suggest that future model versions should use long-term anaesthesia.

Porcine models of non-bacterial thrombotic endocarditis (NBTE) and infective endocarditis (IE) caused by Staphylococcus aureus: a preliminary study.

BACKGROUND AND AIM OF THE STUDY: Non-bacterial thrombotic endocarditis (NBTE) and, in particular, infective endocarditis (IE), are serious and potentially life-threatening diseases. An increasingly important agent of human IE is Staphylococcus aureus, which typically causes an acute endocarditis with high mortality. The study aim was to evaluate the pig as a model for non-bacterial as well as S. aureus-associated endocarditis, as these models would have several advantages compared to other laboratory animal models. METHODS: Fourteen animals underwent surgery with placement of a plastic catheter in the left side of the heart. Six of the pigs did not receive a bacterial inoculation and were used to study the development of NBTE. The remaining eight pigs were inoculated intravenously once or twice with S. aureus, 10(5)-10(7) cfu/kg body weight. Two bacterial strains were used: S54F9 (porcine) and NCTC8325-4 (human). Clinical examination, echocardiography and bacterial blood cultures were used to diagnose and monitor the development of endocarditis. Animals were euthanized at between two and 15 days after catheter placement, and tissue samples were collected for bacteriology and histopathology. RESULTS: Pigs inoculated with 10(7) cfu/kg of S. aureus strain S54F9 developed clinical, echocardiographic and pathologic signs of IE. All other pigs, except one, developed NBTE. Serial blood cultures withdrawn after inoculation were positive in animals with IE, and negative in all other animals. CONCLUSION: S. aureus endocarditis was successfully induced in pigs with an indwelling cardiac catheter after intravenous inoculation of 10(7) cfu/kg of S. aureus strain S54F9. The model simulates typical pathological, clinical and diagnostic features seen in the human disease. Furthermore, NBTE was induced in all but one of the pigs without IE. Thus, the pig model can be used in future studies of the pathogenesis, diagnosis and therapy of NBTE and S. aureus endocarditis.

Organization and biology of the porcine serum amyloid A (SAA) gene cluster: isoform specific responses to bacterial infection.

Serum amyloid A (SAA) is a prominent acute phase protein. Although its biological functions are debated, the wide species distribution of highly homologous SAA proteins and their uniform behavior in response to injury or inflammation in itself suggests a significant role for this protein. The pig is increasingly being used as a model for the study of inflammatory reactions, yet only little is known about how specific SAA genes are regulated in the pig during acute phase responses and other responses induced by pro-inflammatory host mediators. We designed SAA gene specific primers and quantified the gene expression of porcine SAA1, SAA2, SAA3, and SAA4 by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in liver, spleen, and lung tissue from pigs experimentally infected with the Gram-negative swine specific bacterium Actinobacillus pleuropneumoniae, as well as from pigs experimentally infected with the Gram-positive bacterium Staphylococcus aureus. Our results show that: 1) SAA1 may be a pseudogene in pigs; 2) we were able to detect two previously uncharacterized SAA transcripts, namely SAA2 and SAA4, of which the SAA2 transcript is primarily induced in the liver during acute infection and presumably contributes to circulating SAA in pigs; 3) Porcine SAA3 transcription is induced both hepatically and extrahepatically during acute infection, and may be correlated to local organ affection; 4) Hepatic transcription of SAA4 is markedly induced in pigs infected with A. pleuropneumoniae, but only weakly in pigs infected with S. aureus. These results for the first time establish the infection response patterns of the four porcine SAA genes which will be of importance for the use of the pig as a model for human inflammatory responses, e.g. within sepsis, cancer, and obesity research.

Therapy of haematogenous osteomyelitis--a comparative study in a porcine model and Angolan children.

BACKGROUND: It is generally accepted that surgery is necessary for the proper treatment of chronic haematogenous osteomyelitis (HO) in children. However, the correct timing of surgery and the technique most effective for debridement of infectious bone tissue is debated. Theoretically, large animal models of HO can be used for refinement and testing of surgical protocols. We report, to our knowledge for the first time, a porcine model of HO exposed to surgical treatment together with our surgical experiences with Angolan children suffering from chronic HO. MATERIALS AND METHODS: Surgically-debrided bone tissue from the children and pigs were analyzed microbiologically and histopathologically together with the entire operated bones from the pigs. RESULTS: It was illustrated that surgical intervention on porcine bones with experimentally-induced HO is representative of the handling of the condition in children. The porcine HO model can easily be used for refinement and application of surgical techniques used in order to cure children with HO.

A new technique for modeling of hematogenous osteomyelitis in pigs: inoculation into femoral artery.

A new inoculation technique has been developed and applied in a porcine model of juvenile hematogenous osteomyelitis. Following the success of the model, we describe the inoculation technique in detail to enable its replication in future studies. The technique was based on an anatomical feature of the femoral artery that enables inoculation into the artery using a simple surgical procedure. Inoculation in the femoral artery is advantageous because the localization of lesions constitutes a discriminative model of the naturally occurring hematogenous osteomyelitis in long bones, usually involving femur and tibia in children. The procedure was performed under general anesthesia and consisted of five major steps: (1) Exposure of the right femoral artery, (2) retrograde catheterization, (3) inoculation of bacteria, (4) hemostasis of the arterial puncture site using compression, and (5) suturing of the wound in two layers.

Intravenous inoculation of Staphylococcus aureus in pigs induces severe sepsis as indicated by increased hypercoagulability and hepatic dysfunction.

Nine pigs were inoculated intravenously once or twice with 10(8) Staphylococcus aureus per kilogram body weight and sacrificed 12, 24 and 48 h after inoculation. Three sham-infected pigs served as controls. Blood samples were taken for bacteriology, haematology and clinical chemistry. A necropsy was carried out and tissue samples were collected for bacteriology and histology. The onset of clinical disease was seen at 7-8 h after inoculation. The blood bacterial counts remained low throughout the study. All infected pigs developed sepsis characterized by fever, neutrophilia, increased levels of C-reactive protein (CRP) and IL-6, and decreased levels of serum iron. The CRP and IL-6 levels peaked at 36 h, whereas IL-1beta and tumour necrosis factor-alpha showed no obvious changes. Thromboelastography showed increasing hypercoagulability from 12 h and onwards, whereas the platelet numbers declined slightly throughout the experiment. The levels of serum aspartate aminotransferase and bilirubin were elevated at 24 and 36 h. In conclusion, sepsis and severe sepsis were induced as evidenced by dysfunction of the blood clotting system and the liver.

Comparison of B-mode and Doppler ultrasonographic findings with histologic features of benign and malignant superficial lymph nodes in dogs.

OBJECTIVE: To compare and correlate B-mode and color Doppler ultrasonographic characteristics with histopathologic findings of benign and malignant superficial lymph nodes in dogs. STUDY POPULATION: 50 superficial lymph nodes that were normal, abnormally large on physical examination, or represented regional lymph nodes draining an area of suspected primary malignancy in 30 dogs. PROCEDURES: Before excision, lymph nodes were evaluated via B-mode and color Doppler ultrasonography to assess size, echogenicity, presence of a hilus, acoustic transmission, and vascular flow. Formalin-fixed, paraffin-embedded tissue sections of excised lymph nodes were stained with H&E and examined for the presence and extent of necrosis, fibrosis, fat, metastases, and tissue heterogeneity. To assess vascularity, the number and distribution of vessels stained by the Verhoeff van Gieson technique were recorded. RESULTS: In superficial lymph nodes, a varied echogenicity corresponded to tissue heterogeneity. The ultrasonographic detection of a hilus was associated with the presence of fibrous tissue, fat, or both in the hilar region. Acoustic enhancement corresponded to presence of areas of intranodal necrosis. There was significant correlation between both the distribution and the number of vessels detected via ultrasonography and that detected by histopathology. The amount of flow estimated via ultrasonography was typically higher than that estimated via histologic examination. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that histopathologic changes in canine lymph nodes have associated ultrasonographic changes and suggest that lymph node ultrasonography has an important role in the evaluation of lymph nodes in dogs in general and in dogs with neoplastic disease in particular.

Comparison of B-mode and Doppler ultrasonographic findings with histologic features of benign and malignant mammary tumors in dogs.

OBJECTIVE: To compare and correlate B-mode and color Doppler ultrasonographic characteristics with the histologic findings of benign and malignant mammary tumors in dogs. STUDY POPULATION: 49 mammary tumors in 26 dogs. PROCEDURES: Before excision, tumors were evaluated via B-mode and color Doppler ultrasonography to assess size, echogenicity, echopattern, acoustic transmission, invasiveness, and vascularity. Paraffin-embedded microsections of the tumors were stained with H&E and examined for presence of necrosis, cysts, cartilage, bone, mineralization, invasion of surrounding tissue, and tissue heterogeneity. To assess vascularity, the number and distribution of vessels that were stained by the Verhoeff van Gieson technique were recorded. RESULTS: Tumor echogenicity and echopattern on ultrasonographic images correlated with tissue heterogeneity detected histologically. Acoustic enhancement was correlated with the presence of necrotic or cystic areas. Tumor invasion into surrounding tissues as determined ultrasonographically did not correlate with the histologic findings. There was a significant correlation between the number of detected vessels and distribution of flow within the tumors determined via ultrasonographic and histologic examinations. CONCLUSIONS AND CLINICAL RELEVANCE: In canine mammary tumors, ultrasonographic characteristics appear to be correlated with histopathologic changes. Data suggest that ultrasonography may have an important role in the evaluation of mammary tumors in dogs, particularly in the evaluation of tissue composition and tumor vascularity.