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Porcine reproductive and respiratory syndrome virus (PRRSV) remains a leading cause of economic loss in pig farming worldwide. Existing commercial vaccines, all based on modified live or inactivated PRRSV, fail to provide effective immunity against the highly diverse circulating strains of both PRRSV-1 and PRRSV-2. Therefore, there is an urgent need to develop more effective and broadly active PRRSV vaccines. In the absence of neutralizing antibodies, T cells are thought to play a central role in controlling PRRSV infection. Herpesvirus-based vectors are novel vaccine platforms capable of inducing high levels of T cells against encoded heterologous antigens. Therefore, the aim of this study was to assess the immunogenicity and efficacy of an attenuated herpesvirus-based vector (bovine herpesvirus-4; BoHV-4) expressing a fusion protein comprising two well-characterized PRRSV-1 T-cell antigens (M and NSP5). Prime-boost immunization of pigs with BoHV-4 expressing the M and NSP5 fusion protein (vector designated BoHV-4-M-NSP5) induced strong IFN-γ responses, as assessed by ELISpot assays of peripheral blood mononuclear cells (PBMC) stimulated with a pool of peptides representing PRRSV-1 M and NSP5. The responses were closely mirrored by spontaneous IFN-γ release from unstimulated cells, albeit at lower levels. A lower frequency of M and NSP5 specific IFN-γ responding cells was induced following a single dose of BoHV-4-M-NSP5 vector. Restimulation using M and NSP5 peptides from PRRSV-2 demonstrated a high level of cross-reactivity. Vaccination with BoHV-4-M-NSP5 did not affect viral loads in either the blood or lungs following challenge with the two heterologous PRRSV-1 strains. However, the BoHV-4-M-NSP5 prime-boost vaccination showed a marked trend toward reduced lung pathology following PRRSV-1 challenge. The limited effect of T cells on PRRSV-1 viral load was further examined by analyzing local and circulating T-cell responses using intracellular cytokine staining and proliferation assays. The results from this study suggest that vaccine-primed T-cell responses may have helped in the control of PRRSV-1 associated tissue damage, but had a minimal, if any, effect on controlling PRRSV-1 viral loads. Together, these results indicate that future efforts to develop effective PRRSV vaccines should focus on achieving a balanced T-cell and antibody response., Competing Interests: Authors BS-B, MH, HB, and VF are employed by the company ECO Animal Health, London, UK. Authors JR, YW, SH, TM, JS, and MJ in part are employed by the company The Vaccine Group, Plymouth, UK. Author MF was employed by BioVacc Consulting Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that project was funded by ECO Animal Health. The authors declare that this study received funding from ECO Animal Health, who contributed to the design and coordination of the study., (Copyright © 2023 de Brito, Holtham, Roser, Saunders, Wezel, Henderson, Mauch, Sanz-Bernardo, Frossard, Bernard, Lean, Nunez, Gubbins, Suárez, Davison, Francis, Huether, Benchaoui, Salt, Fowler, Jarvis and Graham.)

Background: The effect of a water-soluble formulation of tylvalosin (Aivlosin® 625 mg/g granules) on disease caused by porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae (Mhyop) was investigated in two animal studies. In a PRRSV challenge model in pregnant sows (n = 18), six sows received water medicated at target dose of 5 mg tylvalosin/kg body weight/day from 3 days prior to challenge until the end of gestation. Six sows were left untreated, with a third group remaining untreated and unchallenged. Sows were challenged with PRRSV-2 at approximately 85 days of gestation. Cytokines, viremia, viral shedding, sow reproductive parameters and piglet performance to weaning were evaluated. In a dual infection study (n = 16), piglets were challenged with Mhyop on days 0, 1 and 2, and with PRRSV-1 on day 14 and euthanized on day 24. From day 10 to 20, eight piglets received water medicated at target dose of 20 mg tylvalosin/kg body weight/day and eight piglets were left untreated. Cytokines, viremia, bacteriology and lung lesions were evaluated., Results: In the PRRSV challenge study in pregnant sows, tylvalosin significantly reduced the levels of serum IL-8 (P < 0.001), IL-12 (P = 0.032), TNFα (P < 0.001) and GM-CSF (P = 0.001). IL-8 (P = 0.100) tended to be lower in uterus of tylvalosin sows. All piglets from tylvalosin sows surviving to weaning were PRRSV negative in faecal swabs at weaning compared to 33.3% PRRSV positive piglets from untreated sows (P = 0.08). In the dual challenge study in piglet, tylvalosin reduced serum IL1β, IL-4, IL-6, IL-8, IL-10, IL-12, IL-1α, IL-13, IL-17A, IL-18, GM-CSF, TGFβ1, TNFα, CCL3L1, MIG, PEPCAM-1 (P < 0.001) and increased serum IFNα, IL-1ra and MIP-1b (P < 0.001). In the lungs, tylvalosin reduced IL-8, IL-10 and IL-12 compared to untreated pigs (P < 0.001) and tended to reduce TNFα (P = 0.082). Lung lavage samples from all tylvalosin treated piglets were negative for Mhyop (0 cfu/mL) compared to the untreated piglets which had mean Mhyop counts of 2.68 × 10 4  cfu/mL (P = 0.023)., Conclusion: Overall, tylvalosin reduced both local and systemic proinflammatory cytokines after challenge with respiratory pathogens in sows and in piglets. Tylvalosin was effective in reducing Mhyop recovery from the lungs and may reduce virus shedding in piglets following transplacental PRRSV infection in sows., (© 2023. The Author(s).)

Tumour hypoxia renders cancer cells resistant to cancer therapy, resulting in markedly worse clinical outcomes. To find clinical candidate compounds that reduce hypoxia in tumours, we conduct a high-throughput screen for oxygen consumption rate (OCR) reduction and identify a number of drugs with this property. For this study we focus on the anti-malarial, atovaquone. Atovaquone rapidly decreases the OCR by more than 80% in a wide range of cancer cell lines at pharmacological concentrations. In addition, atovaquone eradicates hypoxia in FaDu, HCT116 and H1299 spheroids. Similarly, it reduces hypoxia in FaDu and HCT116 xenografts in nude mice, and causes a significant tumour growth delay when combined with radiation. Atovaquone is a ubiquinone analogue, and decreases the OCR by inhibiting mitochondrial complex III. We are now undertaking clinical studies to assess whether atovaquone reduces tumour hypoxia in patients, thereby increasing the efficacy of radiotherapy.

Purpose: Profound changes of the vasculature in tumors critically impact drug delivery and therapy response. We aimed at developing a procedure to monitor morphological and functional parameters of the vasculature in subcutaneous xenograft models commonly applied for therapy testing by using probe-based confocal laser endomicroscopy., Procedures: By monitoring various normal and diseased tissues, we established an experimental and analytical set-up to systematically analyze tracer extravasation from the microvasculature. Application of the approach in two xenograft models (HCT-116 and SW620) was realized consecutively throughout tumor growth., Results: The incidence of dilated vessels increased with xenograft size in both models while macromolecule extravasation and tracer accumulation in the tumor tissue, respectively, was significantly reduced throughout growth. The development of dilated/ultradilated vessels correlated with tracer extravasation only in the HCT-116 but not the SW620 model. The underlying mechanisms are still ambiguous and discussed., Conclusions: Our findings clearly indicate that both xenograft type and size matter for drug delivery and therapy testing.

Purpose: Tumour lactate levels have been shown to correlate with high radioresistance in tumour models in vivo. The study aimed to evaluate the impact of pathophysiological extracellular lactate concentrations and acidosis on the in vitro survival and radioresponse of various cancer cell lines., Materials and Methods: HCT-116, HT29 (colorectal) and FaDu (HNSCC) carcinoma cells were studied. Lactate release rates were determined, and expression of the monocarboxylate transporter MCT1 and its cofactor CD147 were monitored by immunofluorescence and flow cytometry. Colony formation was compared for cells exposed to 20 mM exogenous lactate, acidosis (pH 6.4) and lactate plus acidosis relative to control and dose response curves (0.5-10 Gy) were documented., Results: All cell lines expressed MCT1 and CD147 and showed comparable lactate release rates. High lactate levels and acidosis slightly decreased HCT-116 colony forming capacity. This effect was neither additive nor did it affect radioresponse. Clonogenic survival of HT29 cells, however, was critically reduced in a lactate-enriched or acidic milieu and a synergistic effect was observed. Here, both conditions enhanced radiosensitivity. Exogenous lactate also impaired colony formation of FaDu cells but acidosis was ineffective. This cell line was more susceptible to irradiation under lactate exposure independent of pH., Conclusions: Tumour cell behaviour and radioresponse in a lactate environment is multifaceted. The consideration of lactate accumulation as a parameter affecting radiotherapeutic intervention and as a target for new therapeutic strategies is interesting but requires extended mechanistic studies.

Unlabelled: BACKGROUND AND HISTORY: An adult Macaca mulatta was examined because of a history of multiple episodes of conjunctivitis and an acute, pruritic, dermatitic eruption that affected the axillary and inguinal regions, forearms, thorax, and neck., Methods and Results: Results of corneal staining, examination of skin scrapings and feces, fungal culture, CBC, and a thyroid profile (thyroxine/triiodothyronine concentrations) were negative or normal, with the exception of eosinophilia (1,040/mm3). Examination of a punch biopsy specimen of the skin indicated chronic, nonsuppurative eosinophilic dermatitis. Skin patch testing against 25 contact allergens was negative for a delayed-type hypersensitivity reaction. Allergen-specific IgE testing, using six monkey chow additives, also yielded negative results, but testing against latex revealed a strong positive result (0.74 KU/L) consistent with a latex allergy. A skin prick test performed by use of a latex supernatant revealed significant inflammation at the latex site at 72 h and one week. Vinyl gloves were substituted for latex gloves, and that resulted in a marked decrease in erythema, pruritus, and lichenification with no flares of dermatitis for four years. Repeat skin biopsy fourteen weeks after the original biopsy revealed normal epidermis; however, mild chronic active nonsuppurative, perifolliculitis persisted., Conclusion: Latex can induce allergic dermatitis in nonhuman primates and should be included in the differen tial diagnosis for atopic dermatitis.

Background: Because of the uncommon nature of dermal spindle cell tumors, the effectiveness of various treatment modalities is difficult to assess., Objective: Our purpose was to measure the effectiveness of treating dermatofibrosarcoma protuberans, atypical fibroxanthoma, malignant fibrous histiocytoma, and leiomyosarcoma by means of Mohs micrographic surgery (MMS). In addition, we attempted to determine whether MMS is useful in treating dermal spindle cell tumors when no definitive histopathologic diagnosis can be rendered., Methods: In a retrospective chart review, demographic data, tumor data, treatment characteristics, recurrence, and follow-up data were tabulated., Results: The recurrence rate for dermatofibrosarcoma protuberans treated by MMS was 3.0%, for atypical fibroxanthoma was 6.9%, for malignant fibrous histiocytoma was 43%, and for leiomyosarcoma was 14%. The recurrence rate for spindle cell tumors not otherwise specified was 0%., Conclusion: These data establish the effectiveness of MMS in the treatment of dermal spindle cell tumors, including those for which no definitive histopathologic diagnosis can be rendered.

The objective of this work was to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Pasteurella multocida toxin type D, that correlated to a mouse lethality test. Currently, the mouse lethality test is one of several tests used world-wide to evaluate serological responses in animals immunised with vaccines containing toxoids. The mouse lethality test involves injecting mice with a mixture of toxin and test serum sample (from animals that have been vaccinated with a toxoid), and then determining antibody titre of the test serum from the number of mice that survive. Thus, the titre calculated is based on the neutralising activity of the test serum. The mouse lethality test requires large numbers of animals and causes severe distress to the animals. Organisations world-wide are working towards alternatives to animals in the development and control of biological products for human and veterinary use. Additionally, the mouse lethality test is labour-intensive, costly and lacks robustness and may be difficult to reproduce between different technicians. We have developed a double sandwich ELISA to measure anti- P. multocida toxoid type D antibodies in swine serum. Sera from swine immunised with vaccines containing type D toxoid showed good correlation to the mouse lethality assay (Spearman analysis=0.94 and Pearson analysis=0.84). When compared to the mouse lethality test, titres obtained using the ELISA format had higher correlation with protective immunity (i.e., lower turbinate atrophy) following challenge with virulent P. multocida. The ELISA assay is more robust, reproducible and costs less than the mouse lethality assay; and it complements efforts to reduce the use of animals in testing., (Copyright 2001 The International Association for Biologicals.)

Background: Nitroglycerin is a vasodilator that has been reported to improve cutaneous flap and graft survival. It has not been tested in controlled studies., Objective: We designed our study to test the effectiveness of a single postoperative application of nitroglycerin on flap and graft survival., Methods: Eighty-eight surgical repairs received topical nitroglycerin and 85 received control ointment (polysporin). Treatment sites were evaluated on postoperative day 7 and assigned a percentage of surface area survival., Results: There was no significant difference in the complication rate of flaps and grafts treated with nitroglycerin (12.5%) compared with those treated with control ointment (8.4%) (P = .244). Subset analysis of flaps as a group and grafts as a group were not meaningful because the complication rates were so low., Conclusion: There is no survival increase of flaps and grafts treated with a single application of nitroglycerin ointment.

The advent of radiation therapy as a common modality in the treatment and palliation of breast cancer has led to the observation of morphea developing months to years after supervoltage radiation therapy, in and around the site of treatment. We report 2 new cases of morphea at the site of previous supervoltage radiation therapy for breast cancer. The time period between irradiation and onset of morphea in our 2 patients were an atypically long 6.5 years and 32 years, the latter being the longest reported such interval. With conservative treatment, the inflammatory component of the lesions resolved over an approximately 1-year period, leaving residual sclerosis. These patients are compared to those previously reported in the medical literature so as to summarize the range of clinical presentation and course. Recognition of postirradiation morphea is important in distinguishing it from infectious cellulitis, recurrent carcinoma, metastatic carcinoma or development of a second primary carcinoma., (Copyright (R) 2000 S. Karger AG, Basel)

Studies were performed to evaluate the effects of either acute or chronic morphine exposure on histamine release in vivo and supporting studies in vitro. In order to effectively assess histamine release in swine, studies were undertaken to evaluate the effectiveness of compound 48/80 as an intradermal skin test and determine its ability to release histamine in swine cells. Compound 48/80 skin testing was found to be a useful measure of histamine release in swine as evidenced by dose dependent wheal and flare reaction in vivo and histamine release from swine cells in vitro. Acute effects of morphine were determined on swine administered a single injection of morphine alkaloid. Skin tests using intradermal compound 48/80 and histamine, were performed using compound 48/80 both prior to, and 24 h following initiation of morphine treatment. Morphine tolerant swine were subjected to in vivo skin tests and the resulting wheal and flare responses measured. In select swine skin samples from the test sites were measured for mast cell numbers. Swine dermal mast cells were found to release histamine in a dose dependent manner upon compound 48/80 exposure. Both acute and chronic morphine-treated swine had significantly depressed responses to compound 48/80, however this difference was not due alteration in mast cell number or morphology, and skin responsiveness to histamine remained intact.

Clinical trials have shown that currently available commercial vaccines against porcine pleuropneumonia provide inconsistent, serotype-specific protection from the disease. Recovery from naturally acquired infection, however, provides solid, serotype cross-protective immunity. We examined various serum responses of pigs receiving 1 of 4 commercial vaccines or a cell extract, and compared the serologic responses of these pigs after challenge exposure with virulent Actinobacillus pleuropneumoniae serotype 1. Evaluation of serum included complement-mediated killing, opsonizing capacity, IgG titers to whole organisms, and cytotoxin neutralization titers. Pigs that received the cell extract had fewer clinical signs of pleuropneumonia than pigs in other vaccinated groups, and also were significantly (P < 0.05) better protected from development of lung lesions and death. Such vaccinates were the only pigs that developed significant (P < 0.05) serum antibody titers (ie, protective immune response) to whole-cell antigens and to cytotoxin.

Current bacterins provide only partial protection against morbidity and mortality in swine following infection by Actinobacillus pleuropneumoniae. We compared the efficacy of a cell-free concentrate from mid-log phase growth cultures of Actinobacillus pleuropneumoniae (APP) serotype 1 to four commercial bacterins. This cell-free preparation contained carbohydrate, endotoxin, and protein, and had hemolytic and cytotoxic activity. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis analysis indicated the presence of one major 110,000-molecular-weight protein. This protein band also stained by the periodic acid Schiff method, indicating the presence of carbohydrate. Cell-free concentrates of APP serotypes 5 and 7 had identical profiles following electrophoresis and staining with either Coomassie blue for protein or Schiff reagent for carbohydrate. Lipopolysaccharide profiles for the cell-free concentrates of serotypes 1 and 5 were semi-rough while the LPS profile for serotype 7 was smooth. Five A. pleuropneumoniae-free SPF pigs per group were vaccinated on days 0 and 21 with cell-free concentrate of serotype 1 plus adjuvant, or one of four commercial bacterins according to the manufacturer's directions. Control pigs were vaccinated with PBS mixed with adjuvant. All pigs were challenged intranasally on day 35 with serotype 1 and necropsied on day 50. Protection was greatest in the cell-free concentrate group, as compared with all other groups, in that no deaths occurred, clinical scores were less severe, and percent lung affected was significantly reduced (P < 0.05). In addition, whole-cell ELISA titers were significantly increased (P < 0.05) postvaccination in the cell-free concentrate group, and postvaccination and postchallenge sera neutralized the hemolytic activity of the cell-free concentrate from serotypes 1 and 5 (P < 0.05), as compared with all other groups. No serum neutralization to the hemolysin of serotype 7 was observed. Immunoblot analysis using antisera derived from gnotobiotic pigs indicated that the cell-free vaccine generated a response that was identical to the response observed following live challenge. Similar, but not identical, responses were observed when antisera generated against the bacterins was used. This study indicates that an acellular vaccine containing multiple virulence factors can provide complete protection from mortality and significantly reduced morbidity to homologous challenge.

We analysed six multiplex pedigrees of schizophrenia for linkage to two DNA probes mapping to the chromosome 5q11-q13 region where linkage to a gene for schizophrenia was recently reported. Analyses were conducted using three penetrance models and considering the affected state to be schizophrenia, the schizophrenia spectrum, and all psychiatric diagnoses. All analyses gave consistently negative lod scores. Although the region was not formally excluded, no evidence for linkage was found.

Mutants of the unicellular, green alga Chlamydomonas reinhardtii were recovered by screening for the absence of photoautotrophic growth at 35 degrees C. Whereas nonconditional mutants required acetate for growth at both 25 and 35 degrees C, the conditional mutants have normal photoautotrophic growth at 25 degrees C. The conditional mutants consisted of two classes: (a) Temperature-sensitive mutants died under all growth conditions at 35 degrees C, but (b) temperature-sensitive, acetate-requiring mutants were capable of heterotrophic growth at 35 degrees C when supplied with acetate in the dark. The majority of mutants within the latter of these two classes had defects in photosynthetic functions. These defects included altered pigmentation, reduced whole-chain electron-transport activity, reduced ribulosebis-phosphate carboxylase activity, or pleiotropic alterations in a number of these photosynthetic components. Both nuclear and chloroplast mutants were identified, and a correlation between light-sensitive and photosynthesis-deficient phenotypes was observed.