Utility of adenoviral-mediated Fas ligand gene transfer to modulate islet allograft survival

We examined the perioperative balance between oxygen delivery and oxygen consumption under hemodilution in 24 patients who underwent head and neck surgery of long duration and with massive blood loss. Intraoperative mixed venous oxygen saturation (SVO2), which decreased according to a decrease in hematocrit (Hct), was almost always kept above 60% when Hct was more than 20%. The longer the duration of surgery, the lower was SVO2 at the end of surgery. Postoperative SVO2 correlated positively with SVO2 at the end of surgery, and negatively with the duration of surgery and blood loss, but did not correlate with Hct. In 9 cases, SVO2 at the recovery from anesthesia decreased to below 60%, because oxygen consumption increased remarkably with shivering. These results suggest that in highly invasive head and neck surgery, the intraoperative minimum acceptable Hct level is 20%. On the other hand, there is no general postoperative minimum acceptable Hct level, because postoperative oxygen demand and supply balance depends on the degree of surgical invasion and increase of VO2. SVO2 at the end of surgery is useful in predicting postoperative oxygen demand and supply balance.     

In vivo gene transfer methods into bladder without viral vectors

For the application of gene therapy to bladder cancer, we examined four in vivo gene transfer methods without viral vectors. For lipofection cationic liposomes (Lipofectin) were instilled into murine bladders. The hemagglutinating virus of Japan (HVJ)-liposomes possessing membrane fusion activity were also injected intraluminally. Using a particle gun, rabbit bladder mucosa was bombarded with DNA-coated gold microcarriers. Electrotransfection was examined in rabbit bladder by pulse direct currents (0.15-0.2 A, 50 msec, repeated 8 times) generated between needle electrodes after submucous injection of DNA solution. beta-galactosidase gene and chloramphenicol acetyltransferase (CAT) gene were used as marker genes. Although lipofection was inefficient in normal urothelium, cancerous urothelium was transfected slightly. HVJ-liposomes more efficiently transfected superficial layers of urothelium with a peak of expression on day 5. The particle gun produced non-uniform but efficient transfection in deeper layers of the urothelium. By electrotransfection, submucous interstitial cells were transfected as well as urothelium. No major complications were observed after these four procedures. HVJ-liposomes are potentially useful for the treatment of carcinoma in situ and the latter two methods may be suitable for the adjuvant therapy of localized bladder tumors.     

Protection from endotoxemia by adenoviral-mediated gene transfer of human bactericidal/permeability-increasing protein

Sepsis represents a growing concern in high-risk patients and there has been a lack of effective preventives and therapies. Bacterial/permeability increasing protein (BPI) is a human neutrophil granule-associated defense molecule specific for Gram-negative bacteria and their products. To develop a BPI-transgene-based prophylactic or therapeutic modality, we have developed a recombinant, replication-deficient adenoviral vector expressing full-length human BPI protein (AdhBPI). The expression of BPI is under control of a murine cytomegalovirus (CMV) promoter. Using in vitro and in vivo systems, AdhBPI-mediated gene transfer led to extracellular secretion of BPI protein, which effectively neutralized endotoxin (lipopolysaccharide [LPS]) and markedly reduced the production of proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and macrophage inflammatory protein 2 (MIP-2) by freshly isolated murine alveolar macrophages. By using a mouse model of nonlethal sepsis elicited with LPS, we demonstrated that in vivo gene transfer of BPI was able to markedly inhibit the effect of a large dose of LPS on cytokine responses when injected intraperitoneally. Furthermore, such in vivo BPI gene transfer also improved the survival of mice suffering from lethal septic shock elicited by intraperitoneal injection of d-galactosamine and LPS. Thus, our results suggest that human BPI gene transfer vector has the potential to be used as a therapeutic agent for septic conditions.     

In vivo adenoviral vector-mediated gene transfer into balloon-injured rat carotid arteries

Homozygotes for the dsy1 desynaptic mutant of maize show massive failure of chiasma maintenance during diplotene and diakinesis. Although some chiasmata persist until anaphase I in most microsporocytes expressing this mutant, homozygotes are completely or nearly completely sterile, owing apparently to disjunctive irregularities. Pachytene synaptic errors and some synaptic failure also are found, but recombination nodules are common in homologously synapsed regions, and equational separation of a heterozygous knob into univalents or open arms at diakinesis clearly demonstrates that chiasma failure occurs following crossing-over. A wider than normal synaptonemal complex central region and uniform apparent weakness of central region cross connections to spreading procedures strongly suggest the presence of a genetic lesion in a synaptonemal complex central region component. The dsy1 mutant may provide an especially important source of material for molecular studies on the nature of chiasma maintenance mechanism.     

Adenoviral vector-mediated interleukin-10 expression in vivo: intramuscular gene transfer inhibits cytokine responses in endotoxemia

Malacoplakia is a chronic inflammatory disease the etiology of which remains obscure. It has a very low incidence and affects primarily the genitourinary tract, although it has been described in some other organs. This paper presents a historic insight of the clinical cases diagnosed in this centre, and includes a review and update of several issues related to this entity such as pathogenesis, pathological anatomy and treatment. Also, the peculiarities related to the involvement of each separate organ with regard to diagnosis, prognosis and treatment are described.     

In vivo cell lineage analysis during chemical hepatocarcinogenesis using retroviral-mediated gene transfer

We have studied the proliferation of cells in two models of chemical hepatocarcinogenesis. The cells were genetically labeled in vivo using retrovirally mediated transfer of the Escherichia coli beta-galactosidase marker gene coupled to a nuclear localization signal (nls-lacZ gene). In the first carcinogenic model, rats were fed a choline-deficient diet containing 2-acetylaminofluorene, and their livers were perfused with recombinant retrovirus at the onset of oval cell proliferation. The second model was based on the administration of diethylnitrosamine coupled with a partial hepatectomy and is thought to induce cancer with no involvement of oval cells. Analysis of beta-galactosidase expression in the liver at various times after gene transfer revealed the presence of large clusters of positive cells in both models. Moreover, the beta-galactosidase-positive cells displayed morphologic, antigenic, and enzymatic profiles consistent with a hepatocyte phenotype. Our results, therefore, provide evidence for a strikingly similar clonal proliferation of apparently normal hepatocytes during the course of 2-acetylaminofluorene- as well as diethylnitrosamine-induced liver carcinogenesis.     

In vivo gene transfer and expression in rat stomach by submucosal injection of plasmid DNA

We used electromyographic analysis to determine the muscle activity of the shoulder muscles during the lift-off test and during resisted internal rotation. The activity in the upper and lower subscapularis muscle during a lift-off test from the region of the midlumbar spine was approximately 70% of maximal voluntary contraction. This level was significantly higher than for all the other muscles tested (P < 0.05). The lift-off test with the hand placed in the region of the midlumbar spine resulted in one-third more electromyographic activity in the subscapularis muscle than when the test was modified and performed with the hand at the buttocks region. A resisted lift-off test resulted in higher activities in all the muscles, but only a small increase in the pectoralis major muscle. The pectoralis major muscle was significantly more active during resisted internal rotation with the arm in front of the body. Comparison of activity in the upper subscapularis with that in the lower subscapularis muscle showed no significant differences during any of the tests. This study documents the importance of the subscapularis muscle during the lift-off test and suggests that other potential internal rotators of the humerus have a limited role in maintaining internal rotation when the arm is placed behind the back.     

In vivo vascular freezing in clinical microvascular transfer

Free flap transfer to the lower limb in chronic post-traumatic conditions is known to have a higher complication rate, with flap loss in up to 10% of cases, due mainly to the recipient vessels (Godina: Plast Reconstruct Surg 78:285-291, 1986]). The dissection of these vessels often leads to refractory spasm, due to the so-called perivascular post-traumatic disease (PVPTD) (Khouri [Clin Plast Surg 19:773-785, 1992]). A case is presented of the use of a fairly new spasmolytic maneuver, i.e., in vivo vascular freezing, in a patient who developed intractable spasm of the recipient artery.     

Extensive and progressive cutaneous plasmacytoma in plateau phase myeloma

Recently, discussions focused on the question whether acquired activated (APC) resistance is a clue to the observed association between venous thromboembolism (VTE) risk and oral contraceptive (OC) use, especially with the so-called third-generation OC. The objective of our study was to check the validity of acquired APC resistance regarding VTE risk in a case-control study. Sixty-seven women with confirmed VTE diagnosis (n = 67) were consecutively ascertained in primary health care settings, interviewed and blood samples taken (at the earliest 6 months after VTE). Cases were age-matched to 290 population controls. Acquired APC resistance was measured as normalized APC ratio (APCRN). The effect of APC on tissue factor initiated thrombin generation was measured in plasma using alpha 2-macroglobulin attached thrombin activity as an endpoint. Higher risk (odds) ratio with 95% CI) of VTE for carriers of heterozygote Factor V Leiden mutation was confirmed [OR = 2.72 (CI:1.51-4.92)]. However, there is no association between VTE and the level of APCRN OR 0.65 (CI:0.35-1.22). We conclude that acquired APC resistance, measured with a tissue factor initiated test, is unlikely to have a direct association to the clinical outcome of venous thromboembolism.     

In vivo transfer of gene and oligodeoxynucleotides into skin of fetal rats by incubation in amniotic fluid

One of the tools to test an in vitro hypothesis in vivo is transgenic/gene targeting technology. Transgenic technology provides many advantages such as: (1) the study of specific gene function as systemic and developmental effects; and (2) testing of specific gene function chronically. nevertheless, one disadvantage of this technology is the difficulty in tissue-specific targeting of the transgenic expression. Another useful tool is the in vivo gene transfer approach. Therefore, we tested a potential novel model for the study of transgene expression and knock-out using antisense technology. Here, we have demonstrated that incubation of hemagglutinating virus of Japan (HVJ)-liposome complex containing FITC-labeled oligonucleotides in the amniotic fluid of fetal rats resulted in the nuclear localization of fluorescence in the skin (epidermis and dermis). Similarly, transfection of beta-galactosidase gene resulted in positive staining in several surface layers of the skin. Thus, local gene or antisense oligonucleotide transfer approach into the skin may be useful for studying the role of autocrine/paracrine mediators and treating diseases.     

Advance of gene therapy for neurological diseases by direct in vivo gene transfer

Gene therapy for neurological diseases is a rapidly expanding field in neurosciences. It has been demonstrated that some viral vectors from HSV-1 (herpes simplex virus type 1), Ad (adenovirus) and AAV (adeno-associated virus) can transfer foreign genes into nondividing cell types (including neurons). In addition, physical/chemical methods are also used in direct in vivo gene transfer. The direct gene transfer techniques will open a new way to study neurophysiology, neuropathology and therapy for neurological diseases at molecular level. They will hopefully lead to surprising progress in gene therapy for neurological diseases.     

In vivo gene transfer of nitric oxide synthase enhances vasomotor function in carotid arteries from normal and cholesterol-Fed rabbits

BACKGROUND: The vascular endothelium is anatomically intact but functionally abnormal in preatherosclerotic states, and an early deficit in the bioavailability of nitric oxide (NO) or related molecules has been described in both humans and animal models. We hypothesized that the targeted gene transfer of NO synthase (NOS) isoforms might ameliorate or reverse the deficit. METHODS AND RESULTS: We constructed a recombinant adenovirus, Ad.nNOS, that expresses the neuronal isoform of NOS (nNOS) and used it for in vivo endovascular gene transfer to carotid arteries (CA) from normal and cholesterol-fed rabbits. Vessels were harvested 3 days after gene transfer. In CA from normal rabbits, Ad.nNOS generated high levels of functional nNOS protein predominantly in endothelial cells and increased vascular NOS activity by 3.4-fold relative to sham-infected control CA. Ad.nNOS gene transfer also significantly enhanced endothelium-dependent vascular relaxation to acetylcholine; at 3 micromol/L acetylcholine, Ad.nNOS-treated arteries showed an 86+/-4% reduction in precontracted tension, whereas control CA showed a 47+/-6% reduction in tension. Contraction in response to phenylephrine and relaxation in response to nitroprusside were unaffected in both control and Ad.nNOS-treated CA. To determine the effect of Ad.nNOS in atherosclerotic arteries, 10 male New Zealand White rabbits maintained on a 1% cholesterol diet for 10 to 12 weeks underwent gene transfer according to the same protocol used in normal rabbits. Ad.nNOS-treated arteries showed a 2-fold increase in NADPH-diaphorase staining intensity relative to sham-infected and Ad. betaGal-treated arteries. The CA from cholesterol-fed rabbits showed impaired acetylcholine-induced relaxation, but this abnormality was almost entirely corrected by Ad.nNOS gene transfer. CONCLUSIONS: In vivo adenovirus-mediated endovascular delivery of nNOS markedly enhances vascular NOS activity and can favorably influence endothelial physiology in the intact and atherosclerotic vessel wall.