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Direct injection of naked DNA and cytokine transgene expression: implications for keratinocyte gene therapy.

Intradermally injected DNA diffuses into the epidermis and can then enter keratinocytes and become expressed by these cells. Using this method, plasmids containing cytokine genes that have been introduced into keratinocytes can induce a level of cytokine expression sufficient to provide biological effects in the treated skin. Furthermore, transgenic cytokines released from the transduced keratinocytes can also enter the circulation and have downstream effects on other target organs. Thus far, naked DNA injection appears to be a safe, simple, and relatively efficient method that enables genes to be expressed in transplanted human skin on immunosuppressed animals. In humans, keratinocyte gene therapy using the cytokine gene DNA injection method has the potential to become a powerful therapeutic tool for dermatologists in the management of certain inflammatory and other dermatoses.

Biolistics↗

Inhibitory effect of a dendritic epidermal T cell line on K1735 melanoma cells in vivo and in vitro.

Murine epidermis contains a resident population of dendritic epidermal T cells (DETCs) with unknown function. We tested the ability of a cultured line of these cells to inhibit the growth of murine melanoma cells in vitro and in vivo. The interleukin-2 (IL-2)-dependent T cell line AU16, derived from the epidermis of C3H mice, was used as the source of DETCs. AU16 cells were cytotoxic to syngeneic K1735 and CM3205 C3H melanoma cell lines, as well as to a chemically induced cutaneous fibrosarcoma, in an in vitro 51Cr release assay. Mixing viable AU16 cells, but not splenic T cells, with K1735 melanoma cells immediately before intradermal injection into the flank of mice prevented or delayed the outgrowth of melanomas. AU16 cells killed by freezing and thawing and supernatants from cultures of AU16 cells did not inhibit melanoma growth in vivo or in vitro. Injection of viable AU16 cells intravenously or at a separate intradermal site had no effect on melanoma growth. These studies suggest that IL-2-activated DETCs have the potential to inhibit the growth of melanoma cells in vivo and that their cytotoxic activity may be responsible for this effect.

Animals↗

A deviant immune response to viral proteins and transgene product is generated on subretinal administration of adenovirus and adeno-associated virus.

The immune response after ocular exposure to foreign antigens varies substantially from that of a typical systemic response. Anterior chamber associated immune deviation (ACAID) has been well documented. The immune response of the subretinal space has not been studied in as much detail. Here, we characterized the immune response of the subretinal space when it encounters the antigens AdV-GFP and AAV-GFP (recombinant adenovirus or adeno-associated virus, respectively), each delivering the reporter gene encoding green fluorescent protein (GFP). Results indicate that the subretinal space possesses an immune-deviant property similar to ACAID. AdV-elicited immune responses following subretinal injections are significantly reduced compared with systemic responses elicited by intradermal injections of the same virus. Furthermore, subretinal AdV administration results in transduction of retinal pigment epithelial cells (RPE), which are the potential antigen presenting cells of the retina. This subsequently generates a population of immunosuppressive Th2-type, cytokine-secreting, splenic T cells. This response may be advantageous to the development of ocular gene therapy.

Adenoviridae↗

Biological activities of a chemically synthesized form of leukotriene E4.

A chemically synthesized form of leukotriene E4 (LTE4) has been studied for its ability to induce contractions in isolated guinea pig ilea, to induce vascular permeability changes in rat skin when injected intradermally, and to induce bronchoconstriction in guinea pigs after intravenous injection. The synthetic compound induced a contraction in the guinea pig ileum which was slower in developing than that induced by histamine but faster in developing than that induced by a crude preparation of SRS-A isolated from guinea pig lung. The compound was 70-fold more active than histamine on the guinea pig ileum (EC50 of 5 x 10(-9) and 3.5 x 10(-7) M, respectively). FPL 55712, a known SRS-A antagonist, exhibited the same potency in blocking the contractions elicited by the synthetic material as it did in blocking contractions produced by guinea pig SRS-A generated biologically (IC50 of 3.5 x 10(-8) M). The synthetic LTE4 induced a dose dependent increase in vascular permeability in the rat skin which was antagonized by the intravenous injection of FPL 55712 (ID50 of 1.2 mg/kg). The synthetic material was also a potent bronchoconstrictor in the guinea pig when injected intravenously. The bronchoconstriction, too, was antagonized by FPL 55712 when injected intravenously (ID50 of 0.2 mg/kg). In both the rat and guinea pig, FPL 55712 exhibited a short duration of action in vivo. The in vivo model systems discussed in this study, utilizing the synthetic form of LTE4 should be useful in the future evaluation of other SRS-A antagonists.

Animals↗

Adjuvant activity of polymer microparticles and Montanide ISA 720 on immune responses to Plasmodium falciparum MSP2 long synthetic peptides in mice.

The purpose of this work was to test the immunogenicity in C57BL mice of two synthetic peptides derived from the constant region of 3D7 and FC27 Plasmodium falciparum MSP2 dimorphic proteins, either microencapsulated into poly-lactide-co-glycolide acid microparticles (PLGA MP) or delivered with the human compatible adjuvant Montanide ISA 720 for comparison. Potent and prolonged antibody responses were obtained for both peptides by using PLGA MP formulations after subcutaneous or intradermal injections. As compared to the subcutaneous route of immunization, the intradermal route induced greater immune responses. Montanide adjuvant was effective in eliciting antibodies against the 3D7 peptide but not against the FC27 peptide. Peptide-specific cytophilic antibodies (IgG2a) were detected after boosting with homologous peptide for all vaccine formulations. MP formulations elicited a lower IgE secretion as compared to that observed for both Montanide formulated vaccines. Our results demonstrate the ability of the polymer microparticles to overcome the lack of immunogenicity of FC27 MSP2 peptide in C57BL mice and their potential to induce desirable immune responses against malaria.

Adjuvants, Immunologic↗

Success and limitations of a naked plasmid transfection protocol for keratinocyte growth factor-1 to enhance cutaneous wound healing.

Our group and others have previously reported enhancement of cutaneous wound healing following the transfection of tissue with plasmid vectors expressing the DNA for growth factors. In these experiments, growth factor treated animals were usually compared to animals treated with control plasmid vector. To achieve consistent transfection, high DNA plasmid load and repeated penetrations of the wound by needle or gene gun were required. In the current experiments, we assessed the effect of the plasmid load and repeated tissue penetrations on wound healing of excisional wounds in diabetic C57 mice. Animals received 5 mm excisional wounds, and were assigned to the following groups, no treatment, phosphate buffered saline solution injections, and plasmid vector injection with and without the keratinocyte growth factor-1 gene. Intradermal injections of 100 microg plasmid were given adjacent to the wounds at days 1-5, 7 and 11. At day 9, wound closure was more advanced in keratinocyte growth factor-1 treated animals compared to those treated with control plasmid. But a detrimental effect of the DNA plasmid injection was evident from a comparison of the DNA control group versus the non-injected group. Therefore, the challenge for developing an effective system for the enhancement of wound healing lies in improving transfection efficiency.

Animals↗

Role of circulating immune complexes in human secondary syphilis.

Studies in animal models and in the glomerulonephritis of human secondary syphilis and results from in vitro assays have suggested a role for circulating immune complexes (CICs) in human secondary syphilis. Nine adult subjects with early secondary syphilis were studied. All patients tested had CICs on C1q-binding or Raji cell assays. Proteins previously described as Treponema pallidum-specific antigens were detected by radioimmunoblot techniques in CICs from all five subjects tested. Biopsy of early cutaneous lesions revealed immunoreactants (IgG, C3, and/or C1q) in three of nine subjects and treponemal antigen in six of eight subjects tested. Histamine was injected intradermally as a trap for CICs, and biopsy of these injection sites revealed immunoreactants in four of nine subjects and treponemal antigen in five of eight subjects tested. A neutrophilic vascular reaction consistent with CIC-mediated vessel damage was seen in three of nine lesions and six of nine histamine injection sites. Normal controls did not show these changes.

Antigen-Antibody Complex↗

The pathomechanism of posttraumatic edema of lower limbs: I. The effect of extravasated blood, bone marrow cells, and bacterial colonization on tissues, lymphatics, and lymph nodes.

BACKGROUND: The mechanical injury of soft tissues and bones of lower extremities is frequently followed by long-lasting edema at the site of trauma and distally. The pathomechanism of this complication remains unclear. Venous thrombosis and interruption of lymphatics are considered to be the main etiologic factors. We propose a concept that protracted healing of injured tissues and bones with involvement of the regional lymphatic system (lymphatics and nodes) is responsible for persistence of edema. The events affecting the first (scavenging) phase of healing of traumatized tissues, such as hematoma, translocation of bone marrow cells to soft tissues, and colonization by microorganisms, and in particular their effects on lymphatics and lymph nodes, were studied. METHODS: Mongrel dogs weighing 15-20 kg were used. Fresh blood or bone marrow cell (BMC) suspension was injected subcutaneously or intralymphatically into the paw. Strains of saprophyte bacteria residing on the skin surface were cultured and injected intradermally. Oil-contrast lymphography was performed before and after injections to evaluate the changes in lymphatics and nodes. Biopsy samples of paw skin, subcutaneous tissue, and regional lymph nodes (LN) were taken. The responsiveness of LN lymphocytes was studied in autologous mixed cultures with peripheral blood lymphocytes (PBL), BMC, and cultured bacteria. RESULTS: The PBL from subcutaneously injected blood were evacuated by the lymphatic route at a rate of 1-3%/6 hr. There was no thrombosis of lymphatic vessels or obstruction of LN sinusoids. The BMC evoked major inflammatory changes in both the skin and the LN. Bacteria caused local inflammation, dilatation of lymphatics, and destruction of node parenchyma. Autologous BMC and PBL stimulated LN lymphocytes in a 6-day culture. The responsiveness of lymph node lymphocytes to previously subcutaneously injected bacterial antigens was increased. CONCLUSION: The extravasated blood did not produce changes in skin, subcutaneous tissue, and lymphatics; however, it stimulated LN lymphocytes. The BMC and saprophyte bacteria caused major local and lymph node inflammatory response. All these factors may contribute to the local edema in the initial phases of healing of traumatized tissues.

Animals↗

Some inhibitors of histamine-induced and formaldehyde-induced inflammation in mice.

The swelling and capillary hyperpermeability of the mouse foot in response to an injection of formaldehyde, and the increased capillary permeability to an intradermal injection of histamine, have been investigated. Cortisone, mepyramine and sodium salicylate were effective in reducing histamine-induced inflammation, sodium salicylate being less active in the adrenalectomized animal. In formaldehyde-induced inflammation, however, cortisone was ineffective whereas sodium salicylate was effective in the intact mouse, but not in the adrenalectomized animal. Certain aryloxypropionates and anti-esterases were also active in reducing the severity of the formaldehyde-induced inflammation.

Analgesics↗

Studies on leukotriene D4 as an eosinophil chemoattractant.

The effects of LTD4 on eosinophil motility were studied in order to indicate its potential role as an eosinophil chemoattractant. The guinea pig was selected as a suitably sensitive species for in vivo studies. Eosinophil infiltration was quantified by digital image analysis of 6 microns paraffin ocular and cutaneous tissue sections stained by Luna's method. LTD4, applied topically to the ocular surface, caused pronounced eosinophil infiltration into the conjunctival epithelium and was more potent and efficacious than a variety of other putative mediators of allergy. Pretreatment with the LT-antagonist SK&F 104353 (i.v. 1 mg/kg) abolished LTD4-induced eosinophil infiltration into the conjunctiva. Eosinophil infiltration did not occur in other ocular anterior segments structures such as the cornea, iris and ciliary body after either topical application or intracameral injection. LTD4 did not cause eosinophil emigration into skin following intradermal injection, despite causing an increase in cutaneous microvascular permeability at identical doses. These studies indicate that LTD4 may cause eosinophil emigration in vivo according to tissue-dependent regulation.

Administration, Cutaneous↗

Local and systemic reactions in cattle to Hypoderma lineatum larval toxin: protection by phenylbutazone.

Soluble toxin of Hypoderma lineatum injected IV induced a severe cardiorespiratory shock syndrome in calves. Intradermal injection of the toxin caused well-defined, edematous wheals comparable with those produced by histamine. Treatment of calves with famphur (organophosphate insecticide; poured on the skin of the calves 24 hours before injection of toxin) did not increase the severity of the effects of the toxin. Treatment of calves with phenylbutazone (20 mg/kg) 20 minutes before injection of toxin protected the calves against the systemic shock syndrome and the local inflammatory reaction. Apparently, the neurotoxic mechanism of the organophosphate insecticide is independent of the action of the Hypoderma toxin per se. Phenylbutazone can control the effects of the Hypoderma toxin.

Animals↗

Mechanisms of the inflammatory response induced by extracts of Schistosoma mansoni larvae in guinea pig skin.

Penetration of skin by cercariae of Schistosoma mansoni is associated with a local inflammatory response characterised by leukocyte accumulation and tissue swelling. The significance of the inflammation and its relevance to infection of the host is unknown. In this study, we have investigated the mechanisms of the local inflammatory response induced by injecting extracts of Schistosoma mansoni cercariae into guinea pig skin. Intradermal injection of cercarial homogenate or cercarial transformation fluid induced a dose-dependent increase in local edema formation and accumulation of intravenously injected 111In-labeled eosinophils and neutrophils. The responses were rapid in onset and independent of new protein synthesis. The capacity of extracts to induce edema formation and leukocyte accumulation correlated significantly with their proteolytic activity. In addition, extract-induced inflammation was reduced by co-injection with Trasylol, soybean trypsin inhibitor, PMSF, or heparin. A combination of PMSF and heparin virtually abolished extract-induced inflammation. The known inhibitory effect of Trasylol on kallikrein prompted an investigation into the role of kinins in inducing local edema formation. The bradykinin antagonist HOE 140 substantially reduced extract-induced edema whereas bradykinin itself was weak at inducing leukocyte accumulation. The cyclooxygenase inhibitor ibuprofen, the platelet-activating factor antagonist WEB 2086, and the 5-lipoxygenase inhibitor PF5901 had no effect on edema formation. In contrast, extract-induced eosinophil accumulation was reduced by WEB 2086 and PF5901, suggesting that endogenous PAF and leukotriene B4 were involved in recruiting these cells to inflammatory sites. Thus, cercarial proteases induce a local inflammatory response in guinea pig skin that can be attributed, in part, to known mediators of inflammation. It remains to be established whether the response is protective for the host or whether it is beneficial for the parasite. Further understanding of the underlying mechanisms may provide a target for the pharmacologic control of infection in schistosomiasis.

Animals↗

CD 18 monoclonal antibody inhibits neutrophil diapedesis in the murine dermis induced by leukotriene B4 and 12(R)-hydroxyeicosatetraenoic acid.

Neutrophil accumulation is a hallmark of the inflammatory process. The ability of neutrophils to release lipid mediators, toxic oxygen metabolites, proteolytic enzymes and cationic proteins may contribute to the tissue pathology seen in inflammatory diseases such as inflammatory bowel disease and psoriasis. The first step in the process of neutrophil diapedesis in a gradient of chemoattraction is adhesion to the microvascular endothelium, a phenomenon mediated by the stimulated activation of the neutrophil CD11a-c/CD18 cell surface glycoprotein complex. We assessed the ability of a monoclonal antibody (MoAb) (hybridoma: SP2/0-Ag. 14XBALB/c spleen cells; isotype: murine IgG1) to CD18 that recognizes the beta chain of LFA1(CD11a/CD18), MAC-1(CD11b/CD18) and CD11c/CD18 to effect the neutrophils response to the proinflammatory chemotaxins leukotriene B4 (LTB4) and 12(R)-hydroxy-5,8,11,14-eicosatetraenoic acid [12(R)-HETE] in the mouse dermis. LTB4 and 12(R)-HETE induce a time and concentration dependent infiltration of s when applied intradermally. LTB4 (100 ng) and 12(R)-HETE (50 micrograms) were injected intradermally in CD-mice (18 g body weight) and assessed for chemotactic activity four h later by the dermal levels of myeloperoxidase (MPO), a neutrophil marker enzyme. CD18 MoAb(0.02 mg) was given intravenously 10 min ahead of dermal chemotaxin injection. LTB4 increased (p less than .01) dermal levels of MPO at 4 h, a neutrophil accumulation inhibited (p less than .005) by CD18 MoAb pretreatment (Mean MPO +/- SEM: Vehicle, 0.049 +/- 0.006U vs LTB4, 0.309 +/- 0.033U vs MoAb, 0.137 +/- 0.012U) (n = 12/group).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Active immunotherapy of human melanoma exploiting the immunopotentiating effects of cyclophosphamide.

Malignant tumors may escape rejection by the immune system because they induce a state of immunological tolerance mediated by tumor antigen-specific suppressor T cells. In animal systems, cyclophosphamide can reverse the tolerance and thereby facilitate immunologically mediated tumor destruction. We have applied these concepts to the immunotherapy of human malignant melanoma. Forty-three patients with metastatic disease were treated with a whole cell vaccine 3 days after intravenous administration of cyclophosphamide, 300 mg/m2. The vaccine consisted of cryopreserved, irradiated autologous melanoma cells, obtained from metastatic masses by dissociation with collagenase and DNAse, mixed with bacillus Calmette-Guerin (BCG) and injected intradermally. The cyclophosphamide (CY) + vaccine combination was repeated every 28 days. Delayed-type hypersensitivity (DTH) was tested by injecting 1 x 10(6) melanoma cells intradermally and measuring the diameter of induration at 48 h. Most patients had minimal pretreatment DTH responses to melanoma cells (mean +/- SE, mm = 2.4 +/- 0.5). After two vaccine treatments, the responses increased significantly (mean increase +/- SE = 12.1 +/- 1.6 p less than .001) and that level of response was maintained after 4, 6, and 8 treatments. The patients were also skin-tested with a mixture of the enzymes used to dissociate the tumors. No patients exhibited DTH to collagenase + DNAse prior to vaccine injection, but every patient developed DTH to the mixture following two treatments (mean, mm = 26.4 +/- 3.9). Although extracting viable cells from tumor tissue without the use of enzymes proved difficult, we were able to test DTH to mechanically dissociated tumor cells in 23 patients. After two vaccine treatments, there was a significant increase in DTH to enzyme-free autologous melanoma cells (mean DTH +/- SE, mm: 5.4 +/- 1.0, p less than .01). Whereas 5 of 23 patients had positive DTH responses (5 mm induration or greater) before treatment, 11 of 23 were positive after two treatments. Further significant increases in DTH enzyme-free cells were observed after 6 and 8 treatments. Thus, it appears that patients receiving CY + vaccine developed DTH to tumor-associated antigens as well as to residual collagenase and DNAse on the cell surface. Thirty-three patients could be evaluated for antitumor effects of cyclophosphamide + vaccine. There were 3 complete remissions, 1 partial remission, and 2 minor responses. Two complete responders remain alive and free of disease after 57 and 12 months, respectively, and the third died after 39 months. The partial remission consisted of 75% regression of a pulmonary metastasis.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗

Positive relationship of clinical and serologic responses to vaccinia melanoma oncolysate.

In this phase Ia/Ib trial, vaccinia melanoma oncolysate (VMO) is a virus-augmented melanoma cell membrane vaccine that has been shown to be safe and to stimulate the production of antimelanoma antibodies in high-risk melanoma patients treated in a surgical adjuvant setting. One patient with stage I and 38 patients with stage II melanoma were entered in the study between December 1984 and October 1985, with a mean follow-up of approximately 17 months. Each patient received a smallpox booster injection followed one week later by the first of 13 weekly intradermal injections of 2.0 mg of VMO. At the end of 13 weeks, injections were given every other week for 12 months or until recurrence. Clinical results show that 25 of the 39 patients had no evidence of disease as of December 1986. Moreover and more importantly, statistical comparison of patients in this study with 39 matched controls shows a significant increase in disease-free survival for the patients treated with VMO. Serum obtained prior to treatment and at three-month intervals during treatment was tested in a Staphylococcus protein A rosette assay for reactivity with melanoma cell lines. All pretreatment samples (39/39) were negative, and 64% became positive by 12 months after appropriate dosage escalations. Moreover, enzyme-linked immunosorbent assay showed a positive correlation between anti-melanoma IgG antibody titer and disease-free survival.

Adult↗

Investigation of effects of some immunoadjuvants and routes of antigen administration in raising antibodies in rabbits against metacestode cyst antigens of Taenia multiceps.

The relative efficiency of depot (Freund's complete adjuvant (FCA) and saponin), and non-depot (Corynebacterium parvum strain P. acnes I, C. parvum strain IMI585, and levamisole) immunoadjuvants were compared in raising agglutinating and precipitating antibodies against the metacestode fluid of Taenia multiceps of sheep origin (TMCF). Haemagglutinating but not precipitating antibodies were detectable during the period of primary immunisation. Precipitating antibodies of higher titre were produced for longer when TMCF was injected with immunoadjuvant than when the same volume of TMCF was injected alone. Significantly (P less than 0.05) higher indirect haemagglutination (IHA) titres were produced during the primary response following C. parvum treatment than after FCA treatment. FCA treated rabbits showed significantly (P less than 0.05) higher IHA titres than those receiving C. parvum treatment on day 42 only, during the anamnestic response. Ulcers formed at the sites of injection of depot immunoadjuvants. Significantly (P less than 0.05) higher IHA titres were produced when FCA with TMCF was injected intradermally into 10-12 sites than when the same volume was injected into 4 sites. C. parvum strain IM1585 was a better immunoadjuvant in this system than the strain P. acnes I. The intravenous route of administration yielded better titres than the intraperitoneal route when C. parvum strain P. acnes I was used.

Adjuvants, Immunologic↗

Use of hexyl isocyanate antigen to detect antibodies to hexamethylene diisocyanate (HDI) in sensitized guinea pigs and in a sensitized worker.

Hypersensitivity to hexamethylene diisocyanate (HDI) has been reported following occupational exposure. Diagnosis of sensitivity is usually made from clinical evaluation of symptomatology. An in vitro serologic assay for HDI sensitivity was developed by immunizing guinea pigs with HDI and with hexyl isocyanate (HMI). Animals injected intradermally with HMI produced hapten-specific antibodies whereas guinea pigs injected with HDI produced antibodies specific for larger determinants which included the HDI hapten. The larger determinants were assumed to be composed of portions of "self" molecules which reacted in vivo with HDI. Serum albumin appeared to be one such molecule. No cross reactions were noted between antibodies to HDI and another widely used industrial isocyanate, toluene diisocyanate (TDI). Antigens effective in detecting antibodies to HDI or HMI were tested for ability to detect reaginic antibodies in a worker with clinical "HDI" asthma. Using a radioimmunoassay (RAST), antibodies reacted with conjugates containing either HDI or HMI as haptens. In addition, the prevalance of HDI polyisocyanates (Desmodur N) in spray paints prompted its use as a hapten. Antibodies reacted with Desmodur N antigen conjugates in RAST. RAST inhibition further indicated that Desmodur N antigen reacted more readily with the patient's antibodies than did HDI or HMI antigens. These results suggest that the patient may have been exposed to HDI polyisocyanates in spray paint application. Use of Rast inhibition for diagnosis of sensitivity may indicate the precise sensitizing agent within a mixture.

Animals↗

The angiogenesis inhibitor vasostatin does not impair wound healing at tumor-inhibiting doses.

Inhibition of tumor angiogenesis represents a promising new approach for the treatment of human cancers. It has remained unclear, however, whether inhibition of tumor angiogenesis may also result in impaired wound healing, a process thought to be angiogenesis dependent. To determine the effects of the angiogenesis inhibitor vasostatin, a 180 amino acid calreticulin fragment, on wound healing at tumor inhibiting doses, full-thickness wounds were generated on the back of nude mice that were also injected intradermally with CA46 Burkitt lymphoma cells. Mice were treated with daily injections of vasostatin or vehicle control at a site between the wounds and the transplanted tumor cells over 14 d. Vasostatin potently inhibited tumor growth and significantly reduced tumor angiogenesis, as measured by computer-assisted image analysis of CD31-stained tumor sections. Moreover, vasostatin treatment resulted in an increased fraction of mature tumor-associated blood vessels. In contrast, no impairment of wound healing was observed in vasostatin-treated mice, despite a significantly reduced vascularity of the wound granulation tissue. Our results reveal a different sensitivity of malignant tumor growth and physiologic wound healing to inhibition of angiogenesis, and they suggest that therapeutic inhibition of tumor angiogenesis may be achieved without impairment of tissue repair.

Angiogenesis Inhibitors↗