Persistent and secondary adenovirus-mediated hepatic gene expression using adenovirus vector containing CTLA4IgG.

Adenovirus vectors can transfer recombinant genes efficiently into a wide variety of cells in vivo, but have serious limitations: gene expression is transient and secondary gene transfer is inefficient or impossible because of cellular and humoral immune responses against adenovirus-transduced cells. To solve these limitations, we have constructed an adenovirus vector, Adex1CACTLA4IgG, that expresses CTLA4IgG molecules. After in vivo administration of Adex1CACTLA4IgG (9.0 x 10(9) PFU), the peak level of serum CTLA4IgG was 29.8 mg/ml on day 4. The serum CTLA4IgG concentration gradually fell but was still 5.7 mg/ml on day 90. However, the serum concentration of CTLA4IgG was elevated after a second administration of Adex1CACTLA4IgG. The production of antibody against adenovirus was completely prevented after treatment with Adex1CACTLA4IgG. In addition, coadministration of Adex1CALacZ with Adex1CACTLA4IgG induced persistent hepatic expression of beta-Gal molecules, while administration of Adex1CALacZ alone induced transient expression of beta-Gal molecules. More importantly, on day 160 a secondary challenge with Adex1CALacZ was possible in mice treated with Adex1CALacZ plus Adex1CACTLA4IgG. Thus, we have demonstrated that (1) gene expression of a recombinant adenovirus, Adex1CACTLA4IgG, is persistent in liver and secondary administration of this adenovirus is possible, (2) coadministration of Adex1CACTLA4IgG virus with another adenovirus, AdexCALacZ, prolongs AdexCALacZ-mediated gene expression, and (3) Adex1CACTLA4IgG is useful for secondary challenge with Adex1CALacZ.

Successful gene therapy via intraarticular injection of adenovirus vector containing CTLA4IgG in a murine model of type II collagen-induced arthritis.

We previously constructed an adenovirus vector carrying a gene encoding a soluble form of fusion protein, consisting of the extracellular portion of cytotoxic lymphocyte antigen 4 (CTLA4) and the Fc portion of human immunoglobulin G1 (Adex1CACTLA4IgG). Murine type II collagen-induced arthritis (CIA) was treated with Adex1CACTLA4IgG. A single intraarticular injection of 1 x 10(5) PFU was able to support serum CTLA4IgG at more than 10 microg/ml for at least 12 weeks and was able to inhibit the CIA clinically and histologically. In contrast, intravenous, intramuscular, or subcutaneous injection of 1 x 10(5) PFU was unable to support a significant level of serum CTLA4IgG and thus was unable to inhibit the development of arthritis. Thus, we demonstrated that (1) a low-dose intraarticular injection of Adex1CACTLA4IgG was effective in delaying the onset of CIA and reducing the severity of arthritis; (2) an intraarticular (knee joint) injection of Adex1CACTLA4IgG effectively blocked the development of arthritis in distal paws; (3) the inhibitory effect of Adex1CACTLA4IgG lasted at least up to 20 weeks; (4) although serum CTLA4IgG at more than 10 microg/ml persisted for at least 12 weeks, mice treated by intraarticular injection of Adex1CACTLA4IgG were not anergic to adenovirus and were able to mount antibody responses against various antigens.

Reduction in pituitary desensitization and prolongation of gonadotropin release by estrogen during continuous administration of gonadotropin-releasing hormone in women: its antagonism by progesterone.

The present study was designed to elucidate gonadal steroid influences on gonadotropin release and subsequent pituitary desensitization to GnRH. Sixteen women, 10 of whom were normal and 6 of whom had hypogonadism, were infused with GnRH at rates ranging from 0.313-10 micrograms/h via an indwelling iv catheter for 66 h. Blood samples obtained throughout the GnRH infusion were analyzed for LH, FSH, estradiol, and progesterone. A prompt and substantial release of gonadotropin occurred in women with ovarian failure or during the luteal phase in normal women compared with that during the follicular phase of the menstrual cycle. Thereafter, a gradual decrease in gonadotropin secretion occurred due to pituitary desensitization, which was slower in the follicular phase than in other groups. A dose-related increase in integrated LH release occurred during GnRH infusion, but this response tapered off with administration of large doses of GnRH to women with ovarian failure or during the luteal phase. In contrast, it increased linearly up to the maximum dose of GnRH in the follicular phase. These data suggest that 1) basal levels of estrogen suppress the early rapid release of gonadotropin in response to GnRH and reduce subsequent pituitary desensitization, resulting in the prolonged release of LH; 2) estrogen widens the range of dose-related increases in gonadotropin in response to GnRH; and 3) these effects of estrogen are antagonized by progesterone.

Characterization of TSH-positive cells in foetal rat pars tuberalis that fail to express Pit-1 factor and thyroid hormone beta2 receptors.

The thyroid stimulating hormone (TSH)-immunoreactive cells (TSH cells) in the pars tuberalis (PT-TSH cell) of the male rat pituitary gland show an intense spot-like TSH immunoreaction in the paranuclear cytoplasm. However, the ontogenic origin and characteristics of these spot-like stained PT-TSH cells remain to be elucidated. The present study was designed to investigate the distribution and characteristics of PT-TSH cells in the foetal and adult rat pituitary by immunostaining for Pit-1 factor and thyroid hormone receptors (TRs) and reverse transcriptase-polymerase chain reaction (RT-PCR). TSH cells first appeared in the PT at 15.5 days of gestation and were either stained diffusely throughout the cytoplasm or displayed a strongly stained, spotty appearance in the paranuclear region. By 15.5 days of gestation, the rostral part of the PT consisted of columnar epithelium, in which TSH immunoreactivity was spot-like in the apical region of cytoplasm corresponding to the Golgi apparatus. At the 16.5 days of gestation, TSH cells were present in the pars distalis (PD); however, the cells were mostly larger and polygonal with strong staining throughout the cytoplasm. These differences between the PT and PD were retained throughout foetal and neonatal rat development. The TSH cells in the PD of the adult or gestational rat were observed to contain Pit-1 factor by double immunostaining. However, TSH cells in the PT lacked Pit-1 factor. RT-PCR confirmed the absence of Pit-1 and TRbeta2 mRNA in the PT of the adult and late gestation rat pituitary gland. These results suggest that apparently distinct types of TSH cells in the PT develop independently from TSH cells in the PD.

In vitro and in vivo calcification of vascular bioprostheses.

Efficacy of different chemical treatments on calcification of vascular graft in vitro and in vivo was studied. Culture medium-filled rat aortas were separately treated in 0.2% glutaraldehyde and epoxy compound, and photooxidized in 0.01% methylene blue for a shorter period (group 1). Another group of rat aortas were separately treated in the same chemicals for a longer period (group 2). All fresh and treated aortas of both groups were cultured for 21 days in an organ culture medium and implanted (except for group 1) in weanling rats for five months. Histology and immunohistochemistry revealed that differently treated aortas of group 1 grow and calcify, and the smooth muscle cells between elastin fibers are the primary site of calcium deposition. In contrast, differently treated aortas of group 2 neither grew, nor did calcify in the medium except the epoxy compound cross-linked aorta of group 2 which did not grow but did calcify. Untreated aorta did not calcify. All fresh and differently treated aortic homografts calcified severely in rats. Our whole arterial segment-calcification system would be useful for analyzing the molecular and cellular mechanisms of both bioprosthetic and atherosclerotic calcification of vascular graft. New anticalcification technique is the only hope for better outcome of future vascular bioprostheses.

The immature type of pro-opiomelanocortin cell of the rat anterior pituitary as observed by immunogold electron microscopy.

The perinatal development of glandular cells in the rat anterior pituitary producing pro-opiomelanocortin (POMC), the precursor protein of peptide hormones including adrenocorticotropin (ACTH), melanocyte-stimulating hormones (MSH) and endorphins was studied by an immunoelectron microscopic technique using the colloidal gold-antibody method. The POMC cells are classified into the following three types: 1) an immature type, 2) an intermediate type, and 3) a mature type, which correspond to Types III, I and II of the ACTH cells, respectively. The average size of the secretory granules in POMC cells varies widely, measuring 98.6 +/- 8.5 nm in the immature type, 111.4 +/- 6.7 nm in the intermediate type, and 139.3 +/- 23.1 nm in the mature type. The immature type comprises more than 90% of the POMC cells in the late fetal stage, but decreases in number after birth. The intermediate type reaches a peak at 8 days (female) or 33 days (male), while the mature type is that most frequently observed at 45 days, i.e., more than 50-60% of the total POMC cells, when the immature type decreases to about 15%.

Immunocytochemical study using a GABA antiserum for the demonstration of inhibitory neurons in the rat locus ceruleus.

The localization of GABA-like immunoreactivity in the locus ceruleus of rats was studied by the peroxidase-antiperoxidase (PAP) method using a purified antibody raised against GABA applied to paraffin sections, with counterstaining by cresylecht violet, and to floating sections for preembedding immunoelectron microscopy. A few medium-sized and some small neurons showed GABA-like immunoreactivity in both nuclei and perikarya. The preferential localization of these immunopositive neurons in the marginal parts of the locus ceruleus suggests that they are inhibitory local circuit neurons located between this center and the afferent fiber systems. Some of the immunoreactive neurons displayed homogeneous and heterogeneous "paired cells" patterns. Occurrence of the GABA-GABA interaction is indicated. Immunopositive bouton forms are located close to every positive and negative neuron. Electron microscopy confirms GABA-like immunoreactivity in both medium-sized and small neurons of the locus ceruleus and demonstrates that immunoreactive boutons are axosomatic and axosoma spine symmetric synapses on immunopositive and immunonegative cell bodies. These immunocytochemical results support the existence of inhibitory interneurons in the locus ceruleus.

Effects of progesterone on prolactin secretion in hypogonadal women.

Although estrogen is known to stimulate the secretion of prolactin, there are only slight differences between the prolactin levels in the follicular and luteal phases in normal women. To test the hypothesis that progesterone is involved in the regulation of prolactin release, 50 mg of progesterone was administered intramuscularly at 0600 h to twelve hypogonadal women and blood samples were obtained at 15 min intervals between 1500 and 2000 h to determine the prolactin levels. The day before progesterone treatment, control blood samples were obtained at 15 min intervals between 1500 and 2000 h. The serum progesterone levels were 28.7 +/- 4.1 ng/ml at 1500 h, 24.2 +/- 3.5 ng/ml at 1730 h and 21.3 +/- 2.9 ng/ml (mean +/- SD) at 2000 h. In eight of twelve hypogonadal women, progesterone lowered circulating prolactin levels significantly. These results indicate that a high level of progesterone in the luteal phase may partly block estrogen-induced prolactin release physiologically.

Evaluation of GnRH administration on the prolactin response to thyrotrophin-releasing hormone in normal women.

To determine whether GnRH modifies prolactin (PRL) secretion in response to thyrotrophin-releasing hormone (TRH) in normal women, a group of eleven normal women, 23 to 40 years of age, was studied in the mid-follicular phase of the menstrual cycle. The PRL response to TRH was evaluated in serum under control conditions and after GnRH infusion. GnRH administration augmented basal PRL release and amplified TRH-induced PRL release. These results suggest that GnRH may be involved in PRL release, partly by increasing the sensitivity of the lactotrophs to TRH.

Biphasic stimulatory effects of estrogen on gonadotropin surges induced by continuous administration of gonadotropin-releasing hormone in women.

The present experiments were performed to study the effects of preovulatory levels of estrogen on GnRH-induced gonadotropin release. Twelve female volunteers in various phases of the menstrual cycle received estradiol infusion for 66 h at a constant rate of 500 micrograms/24 h which is grossly equivalent to its production rate during the preovulatory follicular phase. In 8 of the women, GnRH was administered concomitantly from 6 h after the initiation of estradiol infusion. The administered doses of GnRH were 2.5 and 5 micrograms/h. Blood samples obtained throughout the infusion were analysed for LH, FSH, estradiol and progesterone. The sole administration of estradiol failed to induce the positive feedback effect on gonadotropin release within the experimental period in the early follicular phase (days 3-7) in 4 women. In 5 women treated during the follicular phase, remarkable LH releases were induced after a lag period by the infusion of both GnRH and estradiol. The induced LH surge formed a prolonged biphasic pattern. Although a similar pattern of FSH was observed in some cases, its response was minimal compared with that of LH. In 3 women during the luteal phase, however, a combined administration of estradiol and GnRH induced only a short term release of LH which was terminated in only 12 h. The present data indicate that 1) Preovulatory levels of estrogen affect the late part of the LH surge which is induced by constant administration of low doses of GnRH resulting in a prolonged biphasic release of LH, and 2) These effects of both hormones are not manifest in the presence of high levels of progesterone. These results indicate the possibility of a role of GnRH and estrogen in the mechanism of the prolonged elevation of a gonadotropin surge at mid-cycle.

Theoretical Debye-Waller factors of alpha-MoO(3) estimated by an equation-of-motion method.

EXAFS oscillations of MoO(3), which has a highly asymmetric local structure, have been calculated using backscattering amplitudes and phase shifts derived from the FEFF8 code and using Debye-Waller factors from an equation-of-motion method. They were compared with polarization-dependent empirical EXAFS data of the alpha-MoO(3) single crystal at various temperatures. The theoretical EXAFS oscillations of Mo-O bonds for the [001] direction of the single crystal, where two symmetric Mo-O bonds exist, reproduced well the experimental data. On the other hand, the calculated data for the [100] direction, which contain two asymmetric Mo-O bonds with different bond lengths, agree well with the experimental data only after adjustment of the amplitude reduction factors for different Mo-O bonds. EXAFS oscillations of MoO(3) powder were also calculated by the same method, and theoretical parameters that could reproduce the experimental data were found. These results suggest that the equation-of-motion method can evaluate the Debye-Waller factors efficiently in molecules with asymmetric local structures and can reduce curve-fitting parameters.

Long-term acceptance of major histocompatibility complex-mismatched cardiac allograft induced by a low dose of CTLA4IgM plus FK506.

The immunosuppressant FK506 prolongs allograft survival. However, at therapeutic doses it has significant side effects. A fusion protein consisting of the extracellular portion of CTLA4 and the Fc portion of human IgG (CTLA4IgG) also prolongs allograft survival, but large doses of CTLA4IgG are required for the induction of cardiac allograft acceptance. Therefore, we constructed a pentameric form of a new CTLA4 fusion protein, CTLA4IgM. We tested whether low doses of CTLA4IgG or CTLA4IgM in combination with subtherapeutic doses of FK506 can prolong allograft survival in a synergistic fashion. C57BL/6 (H-2b) neonatal hearts were transplanted to CBA/J (H-2b) mice in a heterotopic, nonvascularized cardiac allograft model. The findings demonstrate that a combination of low doses of FK506 plus a pentameric form of CTLA4Ig, CTLA4IgM, leads to significant graft survival, while a combination of FK506 plus CTLA4IgG does not.

Hormonal profile after removal of the dominant follicle and corpus luteum in women.

The present experiments were performed to elucidate the mechanism of the selection and maturation of a dominant follicle in women. Eight normally menstruating women undergoing myonectomy or tubal surgery volunteered for the present study. In 3 patients who were operated on Day 9-11, a visual dominant follicle was removed. The other 5 patients underwent the removal of a newly developing corpus luteum on Day 15-21. After taking 3 or 4 preoperative blood samples in the morning after their hospitalization, blood was obtained at 3 or 6 h intervals for the first 36-45 h and at 1-3 day intervals thereafter for 21-34 days. Serum FSH, LH, estradiol and progesterone were measured by radioimmunoassay. Follicleectomy was followed by a sudden drop in estradiol and a minor increase in progesterone. FSH increased for a few days and then declined. There was a drastic, but short-term increase in LH following follicleectomy which was performed before a preovulatory gonadotropin surge. A LH surge occurred 10.7 +/- 1.2 days (mean +/- S.E.) after follicular ablation followed by a luteal phase. In contrast, there was no remarkable LH release in 4 out of 5 patients who underwent luteectomy. A slightly higher level of FSH was sustained for 2-7 postoperative days. "Luteal phase" rises in estradiol and progesterone terminated promptly following luteectomy. A LH surge was observed 14.2 +/- 1.7 days after surgery followed by a luteal phase. After either type of operation, a sustained increase in FSH was followed by a gradual increase in estradiol which preceded a gonadotropin surge. These hormonal sequences resemble those seen in the normal follicular phase. The present data demonstrated that follicleectomy and luteectomy bring on some characteristic hormonal changes which may exert stimulatory or suppressive effects on the selection and maturation of a dominant follicle after the removal of a main ovarian cyclic structure culminating in ovulation at a certain interval.

Follicular development and a single ovulation induced with pulsatile administration of Gn-RH in anovulatory women: studies of hormonal analysis and follicular sonometry.

The method of pulsatile administration of gonadotropin-releasing hormone (Gn-RH) has been proven as a useful means for induction of ovulation in anovulatory women. In our series of clinical trials, 23 out of 29 anovulatory patients ovulated with pulsatile administration of Gn-RH. Seven patients who ovulated volunteered for the present study with daily hormonal analysis and follicular sonometory . Two patients have oligomenorrhea, 3 patients secondary amenorrhea-1st grade (the sole administration of gestagen required for withdrawal bleeding) and the remaining 2 patients secondary amenorrhea-2nd grade (the combined administration of estrogen and gestagen required for withdrawal bleeding). A diagnosis of hyperprolactinemia was made for one patient with secondary amenorrhea-1st grade. Pulsatile administration of Gn-RH was performed by the use of a self-administered infuser . The infuser was connected to an i.v. indwelling catheter via a specially designed blood backflow eliminater . Five micrograms or less of Gn-RH was given every 2 hr from 07:00 to 23:00 hr daily. Five patients received HCG during the preovulatory period. In one patient, a short term treatment of HMG was added to Gn-RH treatment. Follicular sonometry revealed the development of a single dominant follicle which reached between 20 and 28 mm (23.7 +/- 0.12 mm, mean +/- S.E.) in diameter at the preovulatory period. Disappearance of a dominant follicle was recognized in the early luteal phase. Characteristic increases in estradiol were recognized concomitantly with the development of a dominant follicle. Progesterone levels after ovulation were within the limits of its normal "luteal phase" rise. The present data suggest that pulsatile administration of low dose Gn-RH with nocturnal interruption of treatment is effective for normal progress of follicular development in various types of anovulatory patients, culminating in single ovulation. This paper includes the discussion on our method which may be responsible for a high success rate of ovulation induction.

A novel 203 kD aberrant BCR-ABL product in a girl with Philadelphia chromosome positive acute lymphoblastic leukaemia.

We report a girl with Ph1-positive ALL with the aberrant BCR-ABL product. In this case, bcr exon 3 jointed not to ordinal abl exon 2 but to exon 3 resulting in the production of a 203 kD BCR-ABL fusion protein with marked tyrosine kinase activity. To our knowledge, this is the first report of an aberrant BCR-ABL product in childhood. This case was characterized with younger age and low leucocyte count at the onset, but relapsed early like the typical Ph1-positive ALL, suggesting the diversity in the clinicopathogenesis of Ph1-positive ALL.