Development of a recombinant murine tumour model using hepatoma cells expressing hepatitis C virus nonstructural antigens.

Hepatitis C virus (HCV) chronically infects 2%-3% of the world's population, causing liver disease and cancer with prolonged infection. The narrow host range of the virus, being restricted largely to human hepatocytes, has made the development of relevant models to evaluate the efficacy of vaccines a challenge. We have developed a novel approach to accomplish this by generating a murine hepatoma cell line stably expressing nonstructural HCV antigens which can be used in vitro or in vivo to test HCV vaccine efficacies. These HCV-recombinant hepatoma cells formed large solid-mass tumours when implanted into syngeneic mice, allowing us to test candidate HCV vaccines to demonstrate the development of an HCV-specific immune response that limited tumour growth. Using this model, we tested the therapeutic potential of recombinant anti-HCV-specific vaccines based on two fundamentally different attenuated pathogen vaccine systems-attenuated Salmonella and recombinant adenoviral vector based vaccine. While attenuated Salmonella that secreted HCV antigens limited growth of the HCV-recombinant tumours when used in a therapeutic vaccination trial, replication-competent but noninfectious adenovirus expressing nonstructural HCV antigens showed overall greater survival and reduced weight loss compared to non-replicating nondisseminating adenovirus. Our results demonstrate a model with anti-tumour responses to HCV nonstructural (NS) protein antigens and suggest that recombinant vaccine vectors should be explored as a therapeutic strategy for controlling HCV and HCV-associated cancers.

Zinc solubilization and organic acid production by the entomopathogenic fungus, Metarhizium pingshaense sheds light on its key ecological role in the environment.

We report for the first-time higher zinc (Zn) solubilization efficiency and plant growth promotion by an entomopathogenic fungus (EPF), Metarhizium pingshaense IISR-EPF-14, which was earlier isolated from Conogethes punctiferalis, a pest of global importance. The Zn solubilizing efficiency of the fungus varied depending on the type of insoluble source of Zn used, which was observed to be 1.6 times higher in Zn3(PO4)2-amended media compared to ZnO media. In liquid media, there was a 6.2-fold increase in available Zn in ZnO-amended media, whereas a 20.2-fold increase in available Zn was recorded in Zn3(PO4)2 medium. We ascribe the production of various organic acids such as gluconic, keto-gluconic, oxalic, tartaric, malonic, succinic and formic acids, which in general, interact with insoluble Zn sources and make them soluble by forming metal cations and displacing anions as the major mechanism for Zn solubilization by M. pingshaense. However, the type and amount of organic acid produced in the media varied depending on the source of Zn used and the incubation period. Application of the fungus alone and in combination with insoluble Zn sources enhanced various plant growth parameters in rice and cardamom plants. Moreover, the uptake of Zn in rice plants was enhanced up to ~2.5-fold by fungal application. The fungus also exhibited various other plant growth-promoting traits, such as production of Indole-3-acetic acid, ammonia, siderophores, solubilization of mineral phosphate, and production of hydrolytic enzymes such as α-amylase, protease, and pectinase. Hence, apart from its use as a biological control agent, M. pingshaense has the potential to be used as a bio-fortifier to enhance the solubilization and uptake of Zn from nutrient poor soils under field conditions. Our findings shed light on the broader ecological role played by this fungus and widen its scope for utilization in sustainable agriculture.

Exploring Cadaver Skin For Standardization Of Rabbit And Porcine Burn Models In Research.

Burn animal models provide substantial insights into burn pathophysiology. Choice of the apt model is important for determining the clinical efficacy of new medicines. Therefore, standardization of burn models is crucial for scientific research. Use of common techniques like hot water, electricity and incandescent instruments to generate animal burn models is widely reported. However, great discrepancy in employed temperature and exposure times demands user-dependent standardization of the animal model prior to research. Establishment of custom generated in vivo burn models giving consideration to reduced use, suffering and risk of the experimental animal is equally crucial. Accordingly, this pilot study demonstrates a novel approach using rabbit and porcine cadaver skin for standardization of burn parameters prior to use in live animal models. Using a custom-made soldering iron coupled to a 16cm2 surface area copper plate, burns at randomly chosen temperatures of 80˚C and 120˚C, with exposure times ranging from 60s to 180s, were produced on rabbit and porcine cadaver skins. On gross and histopathological analysis, parameters required to generate characteristic changes for deep partial and full thickness burn involvement were established. The identified temperature and exposure time parameters were further validated in live animal models. In vivo validation established the success of this approach, highlighting reduced animal use, ease, reproducibility and efficacy in burn model standardization. The findings of this study will hopefully encourage researchers to opt for cadaver skin to determine parameters required to generate a specific degree of burn prior to its use in live animals for burn research.

Les modèles animaux permettent des avancées substantielles dans la compréhension de la physiopathologie des brûlures. Le choix du modèle est important pour évaluer l'efficacité de nouvelles thérapeutiques, donc la standardisation de ces modèles est cruciale. Les utilisations de l'eau chaude, de l'électricité et de solides chauds est couramment décrite. Cependant, la grande variabilité des températures et des temps de contact nécessite une calibration avant chaque étude expérimentale. Il est en outre nécessaire de développer des modèles in vivo à façon tout en réduisant le risque d'erreur et la souffrance animale. Cette étude décrit l'utilisation de peaux de lapins et de porcs morts pour la standardisation des modèles de brûlure les utilisant. Nous utilisons un fer à souder couplé à une plaque de cuivre de 16 cm², pouvant délivrer une température réglable entre 80 et 120°C pendant 60 à 180 s sur de la peau de lapin ou de porc morts. Nous avons défini les paramètres permettant de réaliser des brûlures intermédiaires ou profondes, objectivées par études macro- et microscopiques, validés ensuite chez l'animal vivant. Cette étape a permis de confirmer l'efficacité de cette approche qui réduit le nombre d'animaux d'expérience, est aisée et reproductible et bien corrélée à l'approche in vivo. Cette étude devrait conduire les équipes a réaliser une calibration ex vivo avant de réaliser une étude in vivo de brûlure expérimentale.

Fever of Unknown Origin in a Renal Transplant Recipient: Lactate Dehydrogenase as an Important Clue to Diagnosis.

Histoplasmosis is a rare disease in nonendemic areas. We report a case of a 23-year-old male patient who presented with fever of unknown origin, cytopenias, organomegaly, and allograft dysfunction 4 months after renal transplant with father as donor. Bone marrow examination showed intracellular budding yeast cells, which was confirmed as histoplasmosis by culture of bone marrow biopsy sample. The patient was treated with intravenous liposomal amphotericin and responded well.

Extracellular matrix compression temporally regulates microvascular angiogenesis.

Mechanical cues influence tissue regeneration, and although vasculature is known to be mechanically sensitive, little is known about the effects of bulk extracellular matrix deformation on the nascent vessel networks found in healing tissues. Previously, we found that dynamic matrix compression in vivo potently regulated revascularization during bone tissue regeneration; however, whether matrix deformations directly regulate angiogenesis remained unknown. Here, we demonstrated that load initiation time, magnitude, and mode all regulate microvascular growth, as well as upstream angiogenic and mechanotransduction signaling pathways. Immediate load initiation inhibited angiogenesis and expression of early sprout tip cell selection genes, while delayed loading enhanced microvascular network formation and upstream signaling pathways. This research provides foundational understanding of how extracellular matrix mechanics regulate angiogenesis and has critical implications for clinical translation of new regenerative medicine therapies and physical rehabilitation strategies designed to enhance revascularization during tissue regeneration.

Regulatory B Cells Are Reduced and Correlate With Disease Activity in Primary Membranous Nephropathy.

INTRODUCTION: Primary membranous nephropathy (PMN) is an autoimmune disease. Both T-regulatory cells (TREGs) and B-regulatory cells (BREGs) are decreased in patients with autoimmune disease. We evaluated the TREG and BREG population in patients of PMN treated with cyclical cyclophosphamide and steroid therapy (cCYC/GC). METHODS: Twenty-four patients with PMN resistant to a restrictive strategy and treated with cCYC/GC therapy and 10 healthy controls were enrolled. The proteinuria, serum creatinine, and serum albumin were tested at monthly intervals and blood samples were collected before starting cCYC/GC and at 6 and 8 (2 months wash out) months of therapy. The peripheral blood mononuclear cells (PBMCs) after staining with fluorochrome-conjugated antibodies were then subjected to flow cytometric analysis for detection of TREGs (CD3+CD4+CD25hiCD127loFoxP3+) and BREGs (CD19+CD5+CD1dhiIL10+). TREGs and BREGs are presented as the percentage of T and B cells, respectively. Cases with remission at month 18 were classified as responders, and those without any remission as nonresponders. RESULTS: Patients with PMN had a lower percentage of TREGs (P = 0.07) and BREGs compared with healthy controls (P = 0.0007). As compared with baseline, there was a significant increase in both BREGs (P = 0.001) and TREGs (P = 0.02) with the treatment (8 months). Patients who responded to therapy by 18 months had an increase in TREG (P = 0.05) and BREG (P = 0.0001) at month 8 compared with baseline. CONCLUSION: As compared with healthy controls, patients with PMN displayed a lower percentage of BREGs. Both TREGs and BREGs significantly improved with disease-specific therapy. BREGs had an association with clinical activity.

Superior immunogenicity of HCV envelope glycoproteins when adjuvanted with cyclic-di-AMP, a STING activator or archaeosomes.

Three decades after the discovery, hepatitis C virus (HCV) is still the leading cause of liver transplantation and poses a major threat to global health. In spite of recent advances in the development of direct acting antivirals, there is still a need for a prophylactic vaccine to limit the virus spread and protect at-risk populations, especially in developing countries, where the cost of the new treatments may severely limit access. The use of recombinant HCV glycoproteins E1E2 (rE1E2) in combination with the MF59, an oil-in-water emulsion-based adjuvant, has previously been shown to reduce the rate of chronicity in chimpanzees and to induce production of cross-neutralizing antibodies and cellular immune responses in human volunteers. To further improve neutralizing antibody responses in recipients along with robust T cell responses, we have explored the immunogenicity of different adjuvants when formulated with the HCV rE1E2 vaccine in mice. Our data show that cyclic di-adenosine monophosphate (c-di-AMP) and archaeosomes elicit strong neutralizing antibodies similar to those elicited using aluminum hydroxide/monophosphoryl lipid A (Alum/monophos. /MPLA) and MF59. However, both c-di-AMP and archaeosomes induced a more robust cellular immune response, which was confirmed by the detection of vaccine-specific poly-functional CD4+ T cells. We conclude that these adjuvants may substantially boost the immunogenicity of our E1E2 vaccine. In addition, our data also indicates that use of a partial or exclusive intranasal immunization regimen may also be feasible using c-di-AMP as adjuvant.

Adult stem cell homing and differentiation in vitro on composite fibrin matrix.

Strategies to generate differentiated cells from haematopoetic progenitor cells will enhance potential use of adult stem cells for therapeutic transplantation or tissue engineering. Transplantation of undifferentiated stem cells into recipient tissue hinges on the hypothesis of a milieu dependent differentiation and it has been suggested that a clot-equivalent scaffold is crucial for these circulating cells to anchor and multiply. Here a natural scaffold, fibrin along with fibronectin, gelatin and growth factors has been used to induce endothelial progenitor cells and smooth muscle progenitor cells to differentiate into endothelial cells and smooth muscle cells, respectively, from peripheral blood mononuclear cells. Characteristics of endothelial cells have been verified by the detection of mRNA for and immunostaining the cells for von Willebrand factor, uptake of acetylated low-density lipoproteins and measurement of released nitric oxide in the culture medium, as nitrite. The specific molecules that characterized smooth muscle cells were alpha smooth muscle actin and calponin, besides deposition of collagen type I and elastin, onto the culture matrix. The adhesive proteins used for the fabrication of endothelial progenitor cells matrix and smooth muscle progenitor cells matrix were the same, but specific differentiation was brought about by modulating the growth factor composition in the matrix and in the culture medium. Both endothelial and smooth muscle cells were consistently developed from 20 ml of human blood.

Deficiency in maturation process of macrophages in human cancer.

Peripheral blood mononuclear cells were cultured from normal individuals and patients with cancer. The analysis of these results indicated a lower number of macrophage precursors (monocytes) in patients with cancer as compared to the number in normal individuals. The macrophage yield was 1.9 +/- 1.1 X 10(4)/ml of blood in patients with cancer versus 7.2 +/- 2.0 X 10(4)/ml of blood in normal individuals. This low yield of macrophages in patients with cancer was not associated with serum factors. Furthermore, a study of the adherent cell population suggests in intrinsic defect in the maturation process in vitro in patients with cancer.

Dose-dependent radiation effect on microvasculature and repair.

It has been postulated that the damage to microvasculature may be a major factor in the manifestation of late radiation damage to organized tissue. In this study, the radiation damage to microvasculature was investigated in a rabbit model during the early phases of irradiation with the use of vascular permeability as a marker. By means of a triple isotopic technique, the vascular, extravascular, and intracellular spaces could be defined. A forelimb and hindlimb of New Zealand White rabbits were irradiated with single doses of 2-30 Gy. 125I- and 131I-labeled bioscreened albumin were used to determine the changes in vascular permeability due to irradiation. The rabbits were sacrificed at various intervals post irradiation. Vascular and extravascular spaces and the respective albumin concentrations in tissue samples from irradiated limbs were compared with control values from the unirradiated contralateral limbs in each animal. The results indicated a definite increase in the vascular permeability of albumin secondary to irradiation. The increase in vascular permeability was apparent instantaneously with irradiation, even at 2 Gy. The microvascular compromise appear to be dose related. When examined at 16-24 hours post irradiation, the excess extravasation of albumin was significant at and beyond 8 Gy. At 10-30 days post irradiation, injury was not apparent up to 15 Gy. Thus there appeared to be an instantaneous injury at the capillary level due to irradiation, which appeared to be dose related. A repair process became evident as early as 16-24 hours and appeared to be dose related as well as related to elapsed time post irradiation.

Recruitment with high physiological doses of estradiol preceding chemotherapy: flow cytometric and therapeutic results in women with locally advanced breast cancers--a Southwest Oncology Group study.

One theoretical method of increasing chemotherapeutic efficacy in breast cancer is to temporarily increase the number of tumor cells in cycle through hormonal recruitment prior to initiation of chemotherapy. In an effort to determine when and if this could be reliably accomplished, 50 women with locally advanced and/or metastatic breast cancer with known estrogen receptor (ER) status were entered into a serial breast biopsy study designed to measure increases in S-phase fraction (SPF) and proliferative index (PI; S + G2 + M) following administration of a high physiological dose of estrogen via estradiol vaginal suppositories prior to chemotherapy. Blood levels of estradiol were maintained in a range (0.5-5 nM) known to increase SPF in vitro. Compliance with suppository administration was monitored by serial blood sampling. Tumors were sampled at 0, 24, 48, 72, and/or 96 h. Thirty-one ER-positive and 9 ER-negative women had evaluable baseline biopsies and at least 1 subsequent biopsy. An increase was seen for SPF in 20 (69%) and for PI in 23 (79%) of 29 ER-positive patients at 48 h after estrogen initiation (95% confidence intervals, 49-85% for SPF and 60-92% for PI); similar increases were seen at 72 h. Median baseline SPF and PI values in ER-positive patients for whom increases were noted at 48 h were 6.2 and 8.5%, respectively. The median relative increases in these patients were 170 and 100%, respectively, at 48 h. The increases observed at 24 h in 4 (SPF) and 6 (PI) of the 9 ER-negative patients could have occurred by chance alone. Twenty-five of the 28 locally advanced (T4 and/or N2-3) patients achieved a complete response during combined modality treatment (estradiol-chemotherapy, mastectomy, and radiation). At a minimum follow-up time of 42 months, estimated 5-year progression-free and overall survivals are 30 and 49%, respectively, with a median time to progression of 35 months. Twenty-two women had metastatic disease (19 also had locally advanced disease). Thirteen had a complete or partial response, with a median duration of 12 months. Median progression-free and over-all survival times for all metastatic patients are 4 and 17 months, respectively. Estimated 5-year survival for metastatic disease patients is 27%. A high physiological dose of estrogen administered to patients with locally advanced ER-positive tumors can reliably increase the tumor SPF and PI within 48 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Archaeal lipid vaccine adjuvants for induction of cell-mediated immunity.

INTRODUCTION: Liposomal vesicles (archaeosomes) composed of total polar lipids (TPL) or semi-synthetic glycerolipids, unique to the domain Archaea, constitute potent vaccine adjuvant and delivery systems. The characteristics of this adjuvant offer a novel prospect for the development of effective vaccines for emerging infections and cancers, which is reviewed in this article. Areas covered: The areas covered in this review include the chemical composition and physical characteristics, various in-vitro and in-vivo pre-clinical immunogenicity and efficacy studies for systemic immunization, induction of mucosal immunity upon modification of the formulation with cations, and the mechanism of adjuvant action following uptake by antigen presenting cells. Expert commentary: The unique features of archaeal lipids confer archaeosomes with many desirable features. With the use of semi-synthetic archaeosomes, highly defined lipids that are safe and robust for induction of cell-mediated immunity may be chosen. These adjuvants function as Toll-like receptor-independent innate immune stimulants.

A structural comparison of the total polar lipids from the human archaea Methanobrevibacter smithii and Methanosphaera stadtmanae and its relevance to the adjuvant activities of their liposomes.

Mice were immunized with bovine serum albumin (BSA) entrapped within archaeosomes (i.e. liposomes) composed of the total polar lipids (TPL) from the two methanogenic archaea common to the human digestive tract. Methanobrevibacter smithii archaeosomes boosted serum anti-BSA antibody to titers comparable to those achieved with Freund's adjuvant, whereas Methanosphaera stadtmanae archaeosomes were relatively poor adjuvants. An explanation for this difference was sought by analysis of the polar lipid composition of each archaeobacterium. Fast atom bombardment mass spectrometry and NMR analyses of the purified lipids revealed a remarkable similarity in the ether lipid structures present in each TPL extract. However, the relative amounts of each lipid species varied dramatically. The phospholipid fraction in M. stadtmanae TPL was dominated by archaetidylinositol (50 mol% of TPL) and the glycolipid fraction by beta-Glcp-(1,6)-beta-Glcp-(1,1)-archaeol (36 mol%), whereas in M. smithii extracts, both caldarchaeol and archaeol lipids containing a phosphoserine head group were relatively abundant. Liposomes prepared from purified archaetidylinositol and from M. stadtmanae TPL supplemented with increasing amounts of phosphatidylserine elicited poor humoral responses to encapsulated BSA. A dramatic loss in the adjuvanticity of M. smithii archaeosomes was seen upon incorporation of 36 mol% of the uncharged lipid diglucosyl archaeol and, to a lesser extent, of 50 mol% of archaetidylinositol. Interestingly, the relative rates of uptake of M. smithii and M. stadtmanae archaeosomes by phagocytic cultures in vitro were similar. Thus, the lipid composition may influence archaeosome adjuvanticity, particularly a high diglucosyl archaeol and/or archaetidyl inositol content, resulting in a low adjuvant activity.

A molecular mechanism for the biphasic effect of exogenous arachidonate on platelets.

A haemoprotein (Mr approximately 40 000) made up of two apparently identical subunits, and having Soret maximum at 405 nm, but displaying it mostly at about 410 nm or often at 415 nm in crude extracts owing to different bound small molecules, found in platelets of several species, has been purified from calf platelets. The purified protein could bind the stable PGH2 analogue, U-46619, without co-operativity (h = 1.0 and half-maximal saturation concentration, S0.5 = 10 microM) and arachidonate with co-operativity that increased with arachidonate concentration (h increasing from approximately 2 to approximately 4). S0.5 of arachidonate was 1.5 microM. Arachidonate binding to the protein was accompanied by its oligomerization.

Conformational responses of an arachidonate- and U46619-binding haemoprotein in relation to platelet activation.

Antibodies were raised, in rabbits, against an arachidonate- and U46619-binding protein purified from calf platelets. Spectral measurements and immunodiffusion experiments were employed to follow conformational responses of the protein in relation to platelet activation. Upon treatment with the platelet agonists, arachidonate and PGH2, as well as the common haem ligands, imidazole and CN-, the purified protein had its Soret band red-shifted, with hypochromicity, but the protein saturated with the agonists, not with the haem ligands, showed altered antigenic properties in immunodiffusion experiments. In an analogous manner activation of gel-filtered calf platelets with high concentrations of ADP and A23187, as well as by cold, had Soret bands of extracts of sonicated platelets red-shifted, with hypochromicity; concomitantly, antigenically different conformations of the protein appeared in Triton X-100 extracts of the activated platelets. A protein immunologically related to the platelet protein was detected in Triton X-100 extracts of calf neutrophils. It is suggested that conformational changes of the protein induced by arachidonate or prostaglandin endoperoxides or H2O2 formed in different compartments during platelet activation by different stimuli may be a biochemical mechanism of stimulus-response coupling and that similar mechanisms might operate in other cell types.

A review of the role of radiation therapy in the treatment of patients with breast cancer.

Irradiation has been used to treat cancer of the breast since 1895. Over the period between 1895 to the present, the therapeutic use of irradiation has evolved from a cauterizing agent to a sophisticated use of theory and practice to cause tumor cell death while at the same time producing maximum normal tissue sparing. The use of radiation has made it possible for the surgeon to use conservative breast sparing surgery to obtain excellent cosmetic results, while at the same time producing a local control equal to that of mastectomy. For the patient, conservative surgery becomes a better option than mastectomy because it not only produces local control equal to that of mastectomy, but gives the wonderful added benefit of a more superior and pleasing cosmetic result.

Immediate effect of irradiation on microvasculature.

The immediate effects of irradiation on microvasculature in muscle in an animal model are described in this paper. By using triple isotopes of 125I, 131I, and 22Na, the transcapillary transfer of albumin from the vascular bed to the extravascular space is determined in terms of mg/g of tissue, after single doses of 2 to 14 Gy. These results reveal an increase in the extravascular albumin immediately after irradiation and suggest an instantaneous compromise in vascular permeability even after 2 Gy. This effect was apparently dose related.

Perioperative interstitial irradiation in the conservative management of early breast cancer.

Conservation of the breast in early breast cancer with limited resection and radiation is proving to be as effective as modified radical mastectomy in survival and in loco-regional control. Management at the University of Kansas Medical Center consists of an interstitial implant at the time of lumpectomy to facilitate perioperative irradiation with Iridium-192 to the tumor bed. An axillary node dissection is also performed at that time. Two to 3 weeks later external beam irradiation is delivered to the entire breast. One hundred and twenty-three breasts in 120 patients have been treated between June 1982 and June 1986. There were 49 pathological Stage I, 63 Stage II, 8 Stage III carcinomas, and 3 carcinomas in situ, consisting of 72 T1, 43 T2, 5 T3, and 3 TIS lesions. Patients have been followed for a median of 30 months. One patient had a "true" recurrence in the breast. Another patient developed recurrence in a different quadrant. Ninety percent of the patients had good to excellent cosmetic results, 7% were considered fair, and 3% had poor results. Seven patients developed mild arm edema, 4 were found to have moderate edema, and 1 had severe arm edema. Our preliminary results indicate that interstitial irradiation immediately after excision results in excellent local control, with very satisfactory cosmesis and no morbidity due to the simultaneous excision and irradiation.

Radiobiological advantages of an immediate interstitial boost dose in conservative treatment of breast cancer.

Minimum surgery with irradiation is emerging as one of the main modalities of therapy for operable early breast cancer. Between June 1982 and June 1986, 110 breasts with Tis, T1 to T3 lesions have been treated at our institution with lumpectomy and interstitial irradiation to the tumor bed with Iridium-192 perioperatively followed by external beam irradiation. There have been two local recurrences at or near the vicinity of the primary, at a median follow-up of 60 months. To analyze the parameters that might have contributed to the local control, we have examined the treatment volumes, prescribed dose to the tumor bed, dose at the core of the tumor bed, and dose to the surrounding normal tissue. Immediate interstitial implant has the radiobiological advantage of delivering continuous low dose irradiation, immediately upon removal of gross tumor to residual foci. Implantation of the afterloading catheters intraoperatively facilitates accurate dose delivery and avoidance of geographical misses. By precise treatment of any residual foci, immediately upon removal of the gross mass, perioperative interstitial irradiation improves local control and by facilitating less radical surgical excision, leads to better cosmetic results.