Construction and molecular characterization of a T-cell receptor-like antibody and CAR-T cells specific for minor histocompatibility antigen HA-1H.

The genetic transfer of T-cell receptors (TCRs) directed toward target antigens into T lymphocytes has been used to generate antitumor T cells efficiently without the need for the in vitro induction and expansion of T cells with cognate specificity. Alternatively, T cells have been gene-modified with a TCR-like antibody or chimeric antigen receptor (CAR). We show that immunization of HLA-A2 transgenic mice with tetramerized recombinant HLA-A2 incorporating HA-1 H minor histocompatibility antigen (mHag) peptides and ß2-microglobulin (HA-1 H/HLA-A2) generate highly specific antibodies. One single-chain variable region moiety (scFv) antibody, #131, demonstrated high affinity (KD=14.9 nM) for the HA-1 H/HLA-A2 complex. Primary human T cells transduced with #131 scFV coupled to CD28 transmembrane and CD3ζ domains were stained with HA-1 H/HLA-A2 tetramers slightly more intensely than a cytotoxic T lymphocyte (CTL) clone specific for endogenously HLA-A2- and HA-1 H-positive cells. Although #131 scFv CAR-T cells required >100-fold higher antigen density to exert cytotoxicity compared with the cognate CTL clone, they could produce inflammatory cytokines against cells expressing HLA-A2 and HA-1 H transgenes. These data implicate that T cells with high-affinity antigen receptors reduce the ability to lyse targets with low-density peptide/MHC complexes (~100 per cell), while they could respond at cytokine production level.

Focal segmental glomerulosclerosis in a case of panhypopituitarism: a possible role of growth hormone treatment.

Panhypopituitarism manifests various symptoms including growth failure, hypothyroidism, adrenal insufficiency and hypogonadism. Dwarfism is an important problem in children with this condition, and long-term treatment with recombinant human growth hormone (GH) is usually required. We report a 24-year-old man with panhypopituitarism complicated by focal segmental glomerulosclerosis (FSGS). The patient had been treated with GH for hypopituitary dwarfism from 3 years of age. Proteinuria was initially noticed at 15 years of age and persisted despite cessation of GH supplementation at 18 years of age. A renal biopsy specimen showed glomerular hypertrophy and limited glomerulosclerosis, compatible with FSGS. To our knowledge, this is the first reported case of panhypopituitarism complicated by FSGS. Our case suggests that GH treatment for dwarfism may induce irreversible glomerular disease.

Regulation of norepinephrine in the medial preoptic area of Siberian hamsters by gonadal steroids.

Photoperiod has profound effects upon the neuroendocrine axis underlying reproductive physiology in seasonally breeding mammals. For long-day (LD) breeders, such as the Siberian hamster, exposure to a short-day (SD) photoperiod results in declines in circulating levels of gonadal steroids, luteinizing hormone (LH), and prolactin (PRL). The current study sought to investigate the effects of photoperiod and steroid levels on norepinephrine (NE), one of the major neurochemical regulators of gonadotropin-releasing hormone (GnRH) function. Since NE release within the medial preoptic area (mPOA) has been shown to stimulate the activity of GnRH cells, it was hypothesized that exposure to a short photoperiod would decrease NE levels. Furthermore, since gonadal steroids show negative feedback on GnRH function, it was hypothesized that gonadectomy would result in increased levels of NE. Adult male and female Siberian hamsters were gonadectomized and implanted with silastic capsules containing either cholesterol (C) or a mixture of estradiol (E) or testosterone (T). Microdialysis sampling within the mPOA was conducted after 8 weeks of exposure to either an LD or an SD photoperiod. Blood samples were analyzed for LH and PRL, while dialysis samples were analyzed for NE and its major metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG). The results revealed significant suppression of LH and PRL by exposure to the SD photoperiod in both males and females. For LH, the steroid implants suppressed circulating hormone levels under both photoperiods, whereas for PRL, steroid treatment facilitated circulating levels. In contrast, there were no significant effects of photoperiod on NE or MHPG release for either males or females, but there was a significant decrease in extracellular levels of these neurochemicals in steroid-treated animals. These data suggest that photoperiodic modulation of GnRH neuronal function by NE is achieved largely through the indirect effects of photoperiod on circulating gonadal steroids.

Hematopoietic progenitor cell counts performed by the Sysmex SE-9000 analyzer can guide timing of peripheral blood stem cell harvest.

BACKGROUND: We studied whether the hematopoietic progenitor cell (HPC) count in peripheral blood as evaluated by an automated counter, the Sysmex SE-9000, correlated with CD34 positive (+) cell count and therefore could guide the timing of peripheral blood stem cell (PBSC) harvest. MATERIALS AND METHODS: HPC count and flow cytometric CD34+ cell count were measured in 90 peripheral blood samples and 30PBSC samples. The correlation between HPC count and apheretic CD34+ cell yield was examined in 19 patients. RESULTS: HPC count showed good correlations with CD34+ cell count in peripheral blood (r = 0.699) and PBSC (r = 0.892). The correlation between peripheral blood HPC count and apheretic CD34+ cell yield also was good (r = 0.789). CONCLUSION: Automated HPC counting can be used as a screening test to guide the timing of PBSC harvest.

[Icteric-type hepatoma with liver cirrhosis successfully treated with hepatectomy after biliary drainage: case report].

A 67-year-old man with liver cirrhosis and an icteric-type hepatoma involving the left main portal vein underwent left hepatic lobectomy after percutaneous transhepatic biliary drainage. Surgery was successful because of effective biliary drainage and meticulous assessment of liver function tests, including 99mTC-galactosyl human serum albumin scintiphotography.

Left hepatic trisegmentectomy for intraductal papillary cholangiocarcinoma: report of a case.

We present a rare case of intraductal papillary cholangiocarcinoma in a 69 year-old man which was treated with left hepatic trisegmentectomy. The hepatic bile ducts were dilated by intraductal masses, which had extended into the intrahepatic bile ducts without involvement of the posterior inferior segmental duct (B6). The patient underwent left hepatic trisegmentectomy with hilar duct resection. The tumors in the posterior superior segmental duct (B7) were resected and biliary reconstruction was performed with a jejunal loop. Post-operative recovery was good, and the patient survived for 7 months after surgery.

Induction of steroidogenic enzyme genes by insulin and IGF-I in cultured adult human adrenocortical cells.

Insulin and the insulin-like growth factors (IGFs) have multiple role in gene expression in steroidogenic cells. We investigated the regulation of steroidogenic enzyme gene expression by insulin and IGF-I in primary cultures of human adrenocortical cells from donors of ages 19-77 years. The effects of insulin and IGF-I observed here were independent of age and sex of the donor. After 5 days in serum-containing medium, cultures were exposed to insulin or IGF-I together with cyclic AMP analogs or ACTH in serum-free defined medium. Insulin and IGF-I at physiological concentrations increased mRNA levels for 17 alpha-hydroxylase and type II 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in the absence of cyclic AMP or ACTH. They had lesser effects on 21-hydroxylase and cholesterol side-chain cleavage enzyme mRNA levels and were3 without effect on 11 beta-hydroxylase mRNA. All steroidogenic enzyme mRNAs were strongly increased by cyclic AMP or ACTH, and this increase was potentiated by insulin or IGF-I. These effects of insulin and IGF-I were accompanied by decreases in the ratio of dehydroepiandrosterone/cortisol synthesized from pregnenolone by the cultures. Induction of steroidogenic enzyme genes in adult human adrenocortical cells by insulin and IGF-I is unlikely to occur by means of a cyclic AMP-dependent mechanism. These data increase the evidence for an important regulation of steroidogenesis by these hormones.

The zona reticularis is the site of biosynthesis of dehydroepiandrosterone and dehydroepiandrosterone sulfate in the adult human adrenal cortex resulting from its low expression of 3 beta-hydroxysteroid dehydrogenase.

Based on indirect evidence, it has often been assumed that the zona reticularis of the adult human adrenal cortex is the source of the adrenal androgens, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS), but direct tests of this concept have been few. Using the techniques of cell culture, Northern blotting, and RIA, we compared the properties of separated adult zonal cells to those of fetal zone cells, a cell type well known to secrete large amounts of DHEA(S) due to its low expression of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD). In nine glands from donors of a wide age range, the zona fasciculata and zona reticularis were separated and dissociated, and the cells were placed in culture. After 5 days, serum was removed by a 24-h period in serum-free defined medium followed by a 24-h exposure to cAMP analogs, with the optional addition of insulin, also in serum-free medium. The separated fasciculata and reticularis cells showed large differences in the DHEA(S)/cortisol (F) production ratios from added pregnenolone precursor, consistent with the synthesis of only F and essentially no DHEA(S) by fasciculata cells and with the synthesis of mostly DHEA(S) with little or no F by both reticularis cells and fetal zone cells. The different patterns of steroidogenesis were accompanied by a much lower level of expression of type II 3 beta HSD in reticularis cells, similar to that in fetal zone cells. In contrast, other genes were similarly regulated in the two adult zones and in the fetal zone by both cAMP and insulin. The levels of messenger ribonucleic acids for 17 alpha-hydroxylase, cholesterol side-chain cleavage enzyme, 21-hydroxylase, and 11 beta-hydroxylase responded to cAMP and insulin in both reticularis cells and fetal zone cells in the same pattern as that previously established in fasciculata cells. The central role of the limited expression of 3 beta HSD in the DHEA(S)-synthesizing property of reticularis cells was established by inhibition of 3 beta HSD in fasciculata cells with trilostane, which caused them to increase their DHEA/F production ratio to a level exceeding even that in fetal zone cells. There did not appear to any age-related changes in gene expression that could account for the large age-related decline in DHEA(S) biosynthesis in humans in either reticularis or fasciculata cells. Thus, the most likely cause of the age-related decline in adrenal androgen biosynthesis is an age-related decline in the number of functional reticularis cells, without a major change in the differentiated properties of the zonal cells as a function of age.

Dual regulation of 21-hydroxylase activity by sex steroid hormones in rat hepatocytes.

In the rat liver, cytochrome P450 catalyzes the hydroxylation of steroid hormones. The expression and activity of some P450 isozymes are regulated by sex steroid hormones. Steroid 21-hydroxylase activity in rat liver is provided mainly by CYP2C6. We studied the regulation of 21-hydroxylase activity by sex steroid hormones in rat primary hepatocyte culture. We added estrogens (estrone, estradiol, estriol) and androgens (testosterone, dihydrotestosterone), (ranging from 10(-9) to 10(-5)M) to the culture. The 21-hydroxylase activity was stimulated by estrogens and was suppressed slightly by androgen in a dose-related manner. The results of our studies demonstrated that sex steroid hormones act differently on 21-hydroxylase activity in rat hepatocytes and, thus, support the hypothesis that the extra-adrenal production of deoxycorticosterone from circulating progesterone is increased during pregnancy by the massive presence of estrogens.

Biphasic regulation by N6,2'-O-dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) of steroid 21-hydroxylase activity in rat hepatocytes.

Steroid 21-hydroxylase activity has been identified in many tissues, including liver. But it is possible that the enzyme found in the liver is different from adrenal 21-hydroxylase. In the adrenal cortex, steroid 21-hydroxylase activity is increased by corticotropin (ACTH); the effect of ACTH is mediated by cyclic AMP (cAMP), and presumably involves a cAMP-dependent protein kinase (PKA). It is not yet clear, however, how extra-adrenal steroid 21-hydroxylase activity is regulated. In the present study, we examined the effect of N6,2'-O-dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP), forskolin, N-[2-(methylamino)ethyl]5-isoquinolinesulfonamide (H-8) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on steroid 21-hydroxylase activity in primary cultures of rat hepatocytes to determine the nature of regulation of extra-adrenal steroid 21-hydroxylase activity. Steroid 21-hydroxylase activity in hepatocytes incubated with 10(-11) M dbcAMP for 24 h was 1.6 times higher than that in control hepatocytes untreated with dbcAMP. On the other hand, steroid 21-hydroxylase activity decreased by 20 and 50% when the cells were incubated with 10(-5) and 10(-3) M dbcAMP, respectively. The stimulatory effect of 10(-11) M dbcAMP was not blocked by 10(-5) M H-8 (PKA inhibitor), but the inhibitory effect of 10(-5) or 10(-3) M cAMP was. TPA did not alter the activity of steroid 21-hydroxylase. These findings indicate that the steroid 21-hydroxylase in rat liver is regulated by mechanisms different from those in the adrenal glands.

A case of Goldenhar syndrome associated with growth hormone deficiency.

We have experienced the case of a 10-year-old boy who had Goldenhar syndrome accompanied by growth hormone (GH) deficiency. His height increased after treatment with growth hormone was administered. We found no untoward effects of the hormone and we consider that treatment with GH is useful for patients who present with Goldenhar syndrome associated with growth hormone deficiency.

Regulation of steroid 21-hydroxylation by 17 beta-estradiol in rat liver: in vivo and in vitro study.

In various extraadrenal organs, progesterone (P4) is converted to deoxycorticosterone (DOC) by steroid 21-hydroxylation. To investigate the regulation of extraadrenal 21-hydroxylase activity by 17 beta-estradiol (E2), the following two experiments were performed. Exp. 1). Three-week-old male SD rats were testectomized (TX) and injected with E2 (1 mg) or sesame-oil s.c. Sham rats were treated with sesame-oil. In these groups the serum concentration of DOC and corticosterone (B), microsomal 21-Hydroxylase activity and the expression of P450c21 mRNA of the liver and the adrenals were analyzed. Exp. 2). Isolated rat hepatocytes were cultured and stimulated by E2, 10(-9) approximately 10(-5) M. 21-Hydroxylase activity of these cells was analysed by the rate of production of DOC in the medium containing P4. The results of experiment 1 showed that both serum DOC concentration and 21-hydroxylase activity in the liver microsomal fraction were increased by E2 injection, but the expression of P450c21 mRNA was not detected in the liver even after E2 injection. In experiment 2, the activity of steroid 21-hydroxylase in isolated rat hepatocytes was stimulated by E2 in a dose dependent manner. These data provided evidence that: in rats the liver was one sites of the extraadrenal steroid 21-hydroxylase activity which had been stimulated by E2. The results also suggested that this hepatic enzyme was a different enzyme from the steroid 21-hydroxylase P450c21 expressed in the adrenal gland.

Effect of anoxic preperfusion on ischemic myocardial injury in isolated rat hearts.

Anoxic perfusion prior to sustained ischemia (anoxic preperfusion), reportedly improves postischemic functional recovery of the heart, but its mechanism has not been well understood. The present study aimed to characterize the cardioprotective effects of anoxic preperfusion and its relationship to extracellular Ca++ levels. Following 10 min of aerobic perfusion, isolated rat hearts were assigned to a 10 min aerobic perfusion or to a 10 min anoxic perfusion. The hearts were then subjected to 30 min of global ischemia and 30 min of aerobic reperfusion. When the perfusate-free Ca++ concentration was 2.0 mM, postischemic recovery of left ventricular developed pressure was significantly improved by anoxic preperfusion (91.9 +/- 2.9% of baseline value vs. 50.5 +/- 12.9% after 30 min reperfusion in the controls). However, the improvement of postischemic ventricular function by anoxic preperfusion was abolished when perfusate Ca++ was reduced to 1.0 mM and the contractile function was rather suppressed during early reperfusion by anoxic preperfusion when the Ca++ level was 0.7 mM (87.5 +/- 11.8% vs. 115.6 +/- 13.9% after 10 min of reperfusion). On the other hand, lactate accumulation during the global ischemia was significantly less in anoxic preperfused hearts compared with untreated hearts both when perfusate Ca++ was 0.7 mM (61.3 +/- 5.1 vs. 85.9 +/- 6.8 mumol/g dry) and when it was 2.0 mM (43.8 +/- 2.0 vs. 140.3 +/- 14.1 mumol/g dry). The amount of myoglobin released after global ischemia was not different between untreated and anoxic preperfused hearts regardless of the perfusate Ca++ level. The results suggest that anoxic preperfusion does not reduce ischemic myocardial necrosis, but it attenuates myocardial stunning. That effect of anoxic preperfusion on the stunning is dependent on the extracellular Ca++ level and is not totally explained by suppression of ischemia-induced lactate accumulation.

[A case of recurrent liver tumor from gastric cancer responding remarkably to hepatic arterial infusion of large doses of mitomycin C and 5-fluorouracil].

A 69-year-old man underwent subtotal gastrectomy for gastric cancer in 1989. He was diagnosed with recurrent liver tumors at 9 months after the operation. Mitomycin C and 5-Fluorouracil were infused repeatedly into hepatic artery, at total doses of 170 mg and 15 g, respectively. Metastatic tumors disappeared in liver by this therapy. There were no side effects such as liver dysfunction, bone marrow suppression and anorexia in spite of the large amount of anti-cancerous agents. This case suggested that hepatic arterial infusion of a large amount of anti-cancerous agents is a good method for liver metastasis from gastric cancer.

Trend analysis of serum progesterone, deoxycorticosterone, deoxycorticosterone sulfate, cortisol, corticosterone, 18-hydroxydeoxycorticosterone and estradiol in early neonates.

To elucidate the origin and regulatory mechanism of deoxycorticosterone (DOC) and deoxycorticosterone sulfate during fetal life, the levels of serum DOC, DOC sulfate, progesterone, cortisol, corticosterone and 18-hydroxydeoxycorticosterone (18OH-DOC) were determined in the fraction separated on high performance liquid chromatogram (HPLC) by radioimmunoassay (RIA) using the serum from normal newborn. Elimination curves both of serum DOC and DOC sulfate showed two phases: rapidly decreasing and slowly decreasing ones. Both serum DOC and DOC sulfate correlated with progesterone (r = 0.340, p less than 0.01; r = 0.737, p less than 0.01, respectively). They also correlated with cortisol (DOC, r = 0.467, p less than 0.01; DOC sulfate, r = 0.549, p less than 0.01, respectively). Serum DOC reached normal adult levels by 16 hrs after birth. However serum DOC sulfate concentration was maintained high throughout the entire early neonatal period. On the contrary, the changes in serum cortisol, corticosterone and 18OH-DOC showed a peak surge in the initial phase after delivery. Both serum corticosterone and 18OH-DOC correlated with cortisol (r = 0.518, p less than 0.01; r = 0.410, p less than 0.01, respectively). These findings suggest that, in the fetus, serum DOC and DOC sulfate are mainly produced at extraadrenal sites isolated from normal mineralocorticoids synthesis and after birth they begin to be formed at adrenal glands.