Anti-apoptotic effect of insulin on normal hepatocytes in vitro and in vivo.

Liver growth factors are known to be anti-apoptotic for hepatocytes. The potential of insulin, a liver co-mitogen, has not been thoroughly evaluated. We studied the anti-apoptotic role of insulin on primary cultures of rat hepatocytes exposed to transforming growth factor-beta (TGF-beta) as the apoptotic agent and in the left portal vein ligation model (LVPL) of liver atrophy. Results show that insulin decreases apoptosis of TGF-beta-treated hepatocyte cultures by 43% (P < 0.002) and the alanine amino transferase (ALT) release by 49% (P < 0.001). Left lobes of LPVL animals displayed a significant increase in the levels of TGF-beta mRNA. In LPVL rats receiving continuous infusion of insulin in the left lobes, the weight of the atrophic lobes was higher over a 7-day period in comparison to control animals. This was associated with lower levels of serum ALT and with a five-fold decrease in the apoptotic index in the left lobes (P < 0.0001). Induction of Akt phosphorylation and increased expression of Bcl-xl were observed in the left lobes of insulin-treated animals. In conclusion, these results show that insulin is anti-apoptotic for normal hepatocytes both in vitro and in vivo and that the action of insulin is associated with increased Bcl-xl expression and Akt activation.

Development of noninvasive and quantitative methodologies for the assessment of chronic ulcers and scars in humans.

Chronic ulcers are a significant and common cause of morbidity and mortality worldwide. They disrupt the epidermis and dermis, resulting in a loss of barrier function. Keloids and hypertrophic scars (benign cutaneous tumors) represent an abnormal healing response. These fibroproliferative disorders are characterized by an overabundance of collagen and accumulation of extracellular matrix due to an imbalance between synthesis and degradation, culminating in excessive scarring. The objectives of this study were to evaluate and compare noninvasive biophysical methods for the measurement of outstanding quantitative parameters of scars and chronic ulcers, and to establish correlations between the parameters measured and the results of conventional subjective clinical evaluations. The development of new technologies, based on ultrasonography and laser Doppler, makes possible new dermatological evaluation methods. Fifteen patients (6 females and 9 males) with 15 chronic ulcers (4 diabetic ulcers, 10 venous ulcers and 1 pressure ulcer) and 30 patients (19 females and 11 males) with 30 scars (25 hypertrophic and 5 keloids) were included in this study. Clinical evaluation was performed by a dermatologist, an aesthetic surgeon and an endocrinologist. Biophysical measurements were used to assess local blood flow by laser Doppler flowmetry (Moor DRT4), thickness and echogenicity by high frequency ultrasonography (20 MHz, Dermascan C) and ulcer linear dimensions by image analysis. Our results show that blood flow within the ulcers and scars was higher than within normal skin. Also, skin thickness of chronic ulcers was decreased when compared to normal skin; the thickness of hypertrophic scars, but not of keloids, was increased in comparison to normal skin, and presented the possibility of measuring wound and scar surfaces with precision. In summary, this pilot study established the feasibility of measuring various biophysical parameters and adapted their potential utility to research on wounds.

Tacrolimus in dermatology.

BACKGROUND: Tacrolimus (FK 506), a metabolite of the fungus Streptomyces tsukubaensis, is an anti-T-cell drug. It acts by inhibiting the production of IL-2, IL-3, IL-4, TNFa, and GM-CSF. More potent and with slightly less secondary effects than cyclosporine, it has been the object of considerable interest, especially in conditions that could benefit from the latter. OBJECTIVE: In psoriasis, a placebo-controlled double-blind study has shown oral tacrolimus at 0.1 mg/kg/day to be effective in controlling recalcitrant lesions. In human, small studies have reported tacrolimus ointment to be effective in controlling acute contact dermatitis. Short-term trials of topical tacrolimus in the treatment of atopic dermatitis have recently shown excellent results in both adults and children. In animal studies of hair growth disorders, topical tacrolimus induces anagen and protects from chemotherapy-induced alopecia. Animal studies with the ointment for the prevention of skin graft rejection, lupus dermatoses, and skin papilloma formation have also shown to be promising. CONCLUSIONS: There are case reports of pyoderma gangrenosum, Sezary's syndrome, and Behcet's disease successfully treated with oral tacrolimus but, because of their small number, they remain anecdotal at this point.

Comprehensive analysis of the acute toxicities induced by systemic administration of cationic lipid:plasmid DNA complexes in mice.

A major limitation associated with systemic administration of cationic lipid:plasmid DNA (pDNA) complexes is the vector toxicity at the doses necessary to produce therapeutically relevant levels of transgene expression. Systematic evaluation of these toxicities has revealed that mice injected intravenously with cationic lipid:pDNA complexes develop significant, dose-dependent hematologic and serologic changes typified by profound leukopenia, thrombocytopenia, and elevated levels of serum transaminases indicative of hepatocellular necrosis. Vector administration also induced a potent inflammatory response characterized by complement activation and the induction of the cytokines IFN-gamma, TNF-alpha, IL-6, and IL-12. These toxicities were found to be transient, resolving with different kinetics to pretreatment levels by 14 days posttreatment. The toxic syndrome observed was independent of the cationic lipid:pDNA ratio, the cationic lipid species, and the level of transgene expression attained. Mechanistic studies determined that neither the complement cascade nor TNF-alpha were key mediators in the development of these characteristic toxicities. Administration of equivalent doses of the individual vector components revealed that cationic liposomes or pDNA alone did not generate the toxic responses observed with cationic lipid:pDNA complexes. Only moderate leukopenia was associated with administration of cationic liposomes or pDNA alone, while only mild thrombocytopenia was noted in pDNA-treated animals. These results establish a panel of objective parameters that can be used to quantify the acute toxicities resulting from systemic administration of cationic lipid:pDNA complexes, which in turn provides a means to compare the therapeutic indices of these vectors.

Reduced inflammatory response to plasmid DNA vectors by elimination and inhibition of immunostimulatory CpG motifs.

An inflammatory response is invariably associated with administration of gene transfer complexes composed of cationic lipids and plasmid DNA (pDNA). In the lung, an influx of neutrophils and elevated levels of several proinflammatory cytokines such as TNF-alpha, IFN-gamma, IL-6, and IL-12 characterize this dose-dependent response. The induction of these cytokines was shown previously to be due in part to the presence of unmethylated CpG dinucleotides in the bacterially derived pDNA. We have eliminated 270 of 526 CpG dinucleotides in a reporter plasmid (pCFA-CAT) and tested the inflammatory response to cationic lipid:pDNA complexes containing the modified vector (pGZA-CAT) after intravenous (i.v.) or intranasal (i.n.) delivery into BALB/c mice. Compared to the unmodified vector, the CpG-reduced pGZA-CAT was found to be significantly less immunostimulatory, as the levels of IL-12, IFN-gamma, and IL-6 in the serum 24 h after i.v. delivery were reduced by 40 to 75%. Similar reductions in cytokine levels were also observed in the bronchoalveolar lavage fluids (BALF) after i.n. administration, while the levels of reporter gene expression were not affected by the modifications. We have also investigated known inhibitors of the CpG signaling pathways in order to decrease the inflammatory response. Two such inhibitors, chloroquine and quinacrine, greatly reduced the induction of IL-12 from mouse spleen cells in vitro and inhibited cytokine production in the lung by approximately 50% without affecting gene expression. These results illustrate that use of a less immunostimulatory pDNA vector or inhibitors of CpG immunostimulation can reduce significantly the toxicity associated with cationic lipid:pDNA complexes thereby increasing the therapeutic index of this synthetic gene transfer vector.

Recombinant human platelet-derived growth factor-BB (becaplermin) for healing chronic lower extremity diabetic ulcers: an open-label clinical evaluation of efficacy.

Topically applied recombinant human platelet-derived growth factor-BB (becaplermin) is a new pharmacologically active therapy for chronic, neuropathic, lower extremity diabetic ulcers. In previous studies, becaplermin gel was administered once daily but dressings were changed twice daily. In the present study of 134 patients with diabetes mellitus and full thickness lower extremity ulcers, dressings were changed only once per day, simplifying the treatment regimen. Efficacy criteria included the percentage of patients achieving complete healing within the 20-week treatment period, the time to achieve complete healing, the rate of ulcer recurrence during the 6-month period following healing, and treatment compliance. Complete healing of ulcers was achieved in 57. 5% of patients, with a mean time to closure of 63 days and a recurrence rate of 21% at 6 months. Of the potential factors affecting ulcer healing, only drug compliance (p < 0.001), dressing compliance (p < 0.01), the presence of infection (p < 0.01), baseline ulcer area (p < 0.05), and baseline total wound evaluation score (p < 0.05) were significantly associated with healing. Results of this study further confirm the efficacy and safety of becaplermin gel for the treatment of lower extremity diabetic ulcers.

[Hypertrichosis and gingival hypertrophy regression in renal transplants following the substitution of cyclosporin by tacrolimus]. / Régression de l'hypertrichose et de l'hypertrophie gingivale chez des greffés rénaux suite à la substitution de la cyclosporine par le tacrolimus.

Gingival Hyperplasia (GH) and hypertrichosis (HT) are two sides effects associated with the usage of cyclosporine (CyA) but not with tacrolimus (FK 506). The aim of this study is to evaluate the efficacy and security of the conversion from CsA to FK 506 to treat those two complications. From August 1996 to May 1997, 15 patients (9 males, 6 females) aged from 23 to 63 years old (38 +/- 14, mean +/- SD) were switched from CsA to FK 506, 12 for GH, 2 for HT and one for combined presentation. FK 506 was first initiated at a dose of 0.15 mg/kg/day and then adjusted to a level target of 8 ng/ml. The conversion was done on an out patient basis at average 35 (5-83) months after transplantation. Patients were followed prospectively for 12 months. There was a significant reduction in GH in all patients within 3 months. Five out 13 patients had a complete resolution of GH within three months of conversion, 9/12 within 6 months and all by 12 months. HT resolved completely within 6 months. No rejection episode occurred and the serum creatinin remain stable over one year post conversion. Conversion from CsA to FK 506 is thus a safe and valid option to treat CsA induced GH and HT.

The role of Apligraf in the treatment of venous leg ulcers.

A cultured, allogeneic, bi-layered human skin equivalent has recently become available to help clinicians manage difficult-to-heal venous ulcers. This skin equivalent has an epidermis and dermis similar to human skin. Its living keratinocytes and fibroblasts are from cultured cell banks derived from human neonatal foreskin. Because the skin equivalent is made up of viable human cells, it cannot be terminally sterilized. Safety concerns, which have been addressed, include the risk of possible transmission of infection, immunogenicity, immunological graft rejection, and tumor formation. However, the maternal blood of the neonatal donor and the master cell banks are screened for infectious agents. Additionally, the human skin equivalent is produced under strict aseptic control, with sterility continuously monitored by the Good Manufacturing Processes. This paper reviews the characteristics of this human skin equivalent and provides practice guidelines.

Binding and uptake of cationic lipid:pDNA complexes by polarized airway epithelial cells.

To better understand the barriers associated with cationic lipid-mediated gene transfer to polarized epithelial cells, Fischer rat thyroid (FRT) cells and polarized normal human bronchial epithelial (NHBE) cells grown on filter supports at an air-liquid interface were used to study the binding and uptake of cationic lipid:plasmid DNA (pDNA) complexes. The efficiencies of binding and uptake of cationic lipid:pDNA complexes by these cell systems were monitored using fluorescence microscopy of fluorescently tagged lipid or pDNA probes. Fluorescent probe bound to the cell surface was differentiated from internalized probe by adding trypan blue, which quenched the fluorescence of bound but not internalized probes. For proliferating cells, binding and internalization of the cationic lipid:pDNA complexes were determined to be efficient. In contrast, little binding or internalization of the complexes was observed using polarized epithelial cells. However, after aspirating a small area of cells from the filter support, virtually all of the cells adjoining this newly formed edge bound and internalized the cationic lipid:pDNA complexes. To determine if their uptake in edge cells was related to the ability of the complexes to access the basolateral membranes of these cells, the binding and uptake of complexes was monitored in polarized NHBE cells that had been pretreated with EGTA or Ca2+-free media, strategies known to disrupt tight junctions. Cells treated in this manner bound and internalized cationic lipid:pDNA complexes efficiently and also expressed significant levels of transgene product. Control cells with intact tight junctions neither bound complexes nor expressed significant transgene product. These data confirm and extend earlier observations that the polarized apical membranes of airway epithelial cells are resistant to transfection by lipid:pDNA complexes. Further, in contrast to previous studies that have shown the entry step of complexes is not an important barrier for COS and HeLa cells, binding and entry of complexes in polarized NHBE cells appear to be rate limiting. These findings suggest that strategies designed to open the tight junctions of polarized epithelial cells may improve gene delivery to these cells for diseases such as cystic fibrosis (CF).

Aerosolization of cationic lipid:pDNA complexes--in vitro optimization of nebulizer parameters for human clinical studies.

Previously, we have described the optimization of the aerosol delivery of a nonviral gene therapy vector to the lungs of rodents (Eastman et al., 1997b). Although aerosolizing cationic lipid:pDNA complexes into a whole-body exposure chamber resulted in high levels of reporter gene expression in the lungs of BALB/c mice, the conditions employed were not optimal for the delivery of lipid:pDNA complexes to the lungs of human patients. That is, the consumption rate of the material in the nebulizer, and thus the delivery time, were very slow and the aerosol was delivered in a continuous flow. Here we describe in vitro experiments used to develop a cationic lipid:pDNA aerosol with characteristics more suitable for delivery to the lungs of humans, as a necessary prerequisite for conducting a clinical study with human cystic fibrosis patients. Using cascade impactors and all-glass impingers, we have screened several commercially available nebulizers for their ability to deliver intact, respirable, active lipid:pDNA complexes in the shortest time possible, and have identified the Pari LC Jet Plus nebulizer as the optimal nebulizer that meets these criteria. Using this nebulizer in an intermittent mode to mimic breath actuation, consumption rates of approximately 0.6 ml/min of the cationic lipid:pDNA complexes (6 mM cationic lipid:8 mM pDNA) were obtained. The plasmid DNA remained intact and the complexes were shown to maintain activity throughout the nebulization run. Based on measurements of the nebulized dose and the mass median aerodynamic diameter, we calculate a delivered dose of approximately 22 micromol (7.2 mg) of pDNA for each 8 ml of cationic lipid:pDNA complex aerosolized to the lungs of a human patient. This dose should be sufficient to test the clinical efficacy of cationic lipid-mediated gene delivery for the treatment of cystic fibrosis.

Biophysical characterization of cationic lipid: DNA complexes.

To better understand the structures formed by the interaction of cationic lipids with DNA, we undertook a systematic analysis to determine the biophysical characteristics of cationic lipid:DNA complexes. Four model cationic lipids with different net cationic charge were found to interact in similar ways with DNA when that interaction was compared in terms of the apparent molar charge ratio of lipid to DNA. When DNA was present in charge excess over the cationic lipid, the complex carried a net negative charge as determined by zeta potential measurements. Under these conditions, some DNA was accessible to ethidium bromide, and free DNA was observed in agarose gels and in dextran density gradients. Between a lipid:DNA charge ratio of 1.25 and 1.5:1, all the DNA became complexed to cationic lipid, as evidenced by its inaccessibility to EtBr and its complete association with lipid upon agarose gel electrophoresis and density gradient separations. These complexes carried a net positive charge. The transition between negatively and positively charged complexes a occurred over a very small range of lipid to DNA ratios. Employing a fluorescent lipid probe, the addition of DNA was shown to induce lipid mixing between cationic lipid-containing vesicles. The extent of DNA-induced lipid mixing reached a maximum at a charge ratio of about 1.5:1, the point at which all the DNA was involved in a complex and the complex became positively charged. Together with freeze-fracture electron micrographs of the complexes, these biophysical data have been interpreted in light of the existing models of cationic lipid:DNA complexes.

A concentrated and stable aerosol formulation of cationic lipid:DNA complexes giving high-level gene expression in mouse lung.

Advances in gene therapy vectors and techniques hold promise for treatment of many inherited and acquired diseases. For lung indications, especially those involving the epithelium, delivery of the gene therapy vehicle ideally will involve the use of an aerosol. Aerosol delivery of transgenes using cationic lipids is currently limited by the ability to generate highly concentrated formulations of lipid:DNA complexes that are stable and retain their activity following aerosolization. We have examined many of the variables inherent in aerosolizing cationic lipid gene delivery vehicles and have devised a new formulation that incorporates small amounts of a polyethylene glycol-containing lipid. This formulation has allowed the preparation of concentrated dispersions of cationic lipid:plasmid DNA (pDNA) complexes (> 20 mM pDNA) at approximately 10-fold higher concentrations than previously reported. Most of the pDNA in these formulations was bound to the lipid component and thereby protected from nebulizer-induced shearing; the pDNA also maintained full biological activity both in vitro and in vivo. This new formulation thus represents a significant improvement over current methods to prepare concentrated, active cationic lipid gene delivery vectors, and provides a new tool with which to test gene transfer to the lung.

Optimization of formulations and conditions for the aerosol delivery of functional cationic lipid:DNA complexes.

We have examined several variables inherent in aerosolizing cationic lipid:DNA complexes using a jet nebulizer and thereby have optimized the delivery of functional complexes. Maximal aerosol transfer efficiency of cationic lipid:pDNA complexes was quantitated and shown to require the presence of at least 25 mM NaCL as an excipient. This is possibly related to effects on the measured zeta potentials of the complex, which indicate that the complexes are more highly charged in solutions of physiological ionic strength than in solutions of low ionic strength. Inclusion of saline also resulted in retention of the starting lipid to plasmid DNA (pDNA) ratio following nebulization. These data were used to design in vitro aerosolization experiments with tissue culture cells that resulted in the identification of a cationic lipid:pDNA ratio of 0.75:1 (mol:mol) as being optimal for aerosolization. This formulation largely protected pDNA from shear degradation during nebulization and produced a respirable aerosol droplet size (1-3 microns). It was tested further in a mouse model and shown to result in the dose-dependent transfection of mouse lungs, generating the equivalent of several picograms of reporter gene activity per mouse lung. The results of these experiments have provided a set of optimal conditions for nebulizing cationic lipid:pDNA complexes that can be used as a starting point for the further evaluation of aerosol delivery of these nonviral gene delivery vectors in vivo.

Detection of a transforming fragment of herpes simplex virus type 2 in clinical specimens by PCR. The Canadian Women's HIV Study Group.

A PCR assay for the sensitive detection of a transforming fragment of herpes simplex virus type 2 (HSV-2) was developed. Oligonucleotide primers were selected in Xho-2, a transforming region of the BglII N fragment of HSV-2. The assay reached a sensitivity endpoint of 10 copies of the Xho-2 subfragment and did not show cross-reactivity with other herpesviruses, including HSV-1. All 42 HSV-2 isolates scored positive in the assay. The Xho-2 PCR assay was evaluated with 216 clinical specimens and the results were compared with those of cell culture. The best protocol for processing specimens contained in viral transport medium included a centrifugation step followed by cell lysis. Of the 107 specimens positive for HSV-2 by culture, 105 were PCR positive (sensitivity, 98.1%). For one of the two falsely negative samples, beta-globin as well as sequences from the HSV-2 DNA polymerase gene could not be amplified. The other sample scored positive in both of these reactions but was indeterminate in duplicate tests by Xho-2 PCR. Two of 109 HSV-2 culture-negative specimens tested positive in the PCR assay (specificity, 98.2%). The latter two samples tested positive in a PCR test for the HSV-2 DNA polymerase gene. This novel tool was shown to be sensitive and specific for HSV-2 sequences and should allow for the investigation of the role of HSV-2 in genital cancers.