Action of neuropeptide Y on nociceptive transmission in substantia gelatinosa of the adult rat spinal dorsal horn.

Effects of neuropeptide Y (NPY) on substantia gelatinosa neurons were investigated in adult rat spinal cord slices using blind whole-cell patch-clamp technique. Bath application of NPY (1 microM) induced a membrane hyperpolarization, resulting in a suppression of the dorsal root stimulation-induced action potentials in 24% of the substantia gelatinosa neurons tested. In voltage clamp mode, NPY produced an outward current dose-dependently in about one third of substantia gelatinosa neurons at the holding potential of -60 mV, which was not affected by tetrodotoxin (1 microM). The NPY-induced current was suppressed by perfusion with a Ba2+-containing external solution and a Cs2SO4 or tetraethylammonium-containing pipette solution. In addition, The NPY-induced outward currents reversed its polarity near the equilibrium potential of K+ ions (-93 mV). The response to NPY recorded with guanosine-5'-O-(2-thiodiphosphate)-beta-S (GDP-beta-S) containing pipette solution was abolished 30 min after patch formation, suggesting that the response was mediated by the G-protein-coupled receptors. Application of an NPY-Y1 selective agonist, [Leu(31), Pro(-34)]-NPY (1 microM), for 30 s also induced an outward current with a similar time course and amplitude to that induced by NPY. On the other hand, the NPY response was blocked by a simultaneous application of NPY-Y1 selective antagonist, BIBP 3226 (1 microM). No significant changes were found in amplitude and frequency of miniature excitatory postsynaptic currents and dorsal root evoked excitatory postsynaptic currents by NPY. In addition, NPY did not affect both of the miniature inhibitory postsynaptic currents and evoked inhibitory postsynaptic currents, mediated by either the GABA or glycine receptor. These findings, taken together, suggest that NPY produces an outward current in substantia gelatinosa neurons through G-protein coupled, and NPY-Y1 receptor-mediated activation of K+ channels without affecting presynaptic components. The inhibition of the synaptic transmission from the primary fibers to the substantia gelatinosa neurons is considered to contribute to the antinociceptive effects of NPY.

Prolonged effects of polyriboinosinic:polyribocytidylic acid on spontaneous running wheel activity and brain interferon-alpha mRNA in rats: a model for immunologically induced fatigue.

Following 2 weeks acclimation to the running wheel in the home cages, an i.p. injection of a synthetic double-stranded RNA, polyriboinosinic:polyribocytidylic acid (poly I:C, 3 mg/kg), was performed to produce the immunologically induced fatigue in rats. The daily amounts of spontaneous running wheel activity decreased to about 40-60% of the preinjection level until day 9 with normal circadian rhythm, then gradually returned to the baseline level by day 14. Rats given a heat exposure (36 degrees C for 1 h) for the consecutive 3 days showed an increase in activity except for the first day. In the open field test, the total moving distance and the number of rearing of the poly I:C-injected rats decreased on day 1, but they were not different from the saline-injected group on day 7, suggesting that the poly I:C-induced fatigue on day 7 was not due to the peripheral problems such as muscle/joint pain, but involved the CNS. Quantitative analysis of mRNA levels using a real-time capillary reverse transcriptase-polymerase chain reaction (RT-PCR) method revealed that interferon-alpha (IFN-alpha) mRNA contents in the cortex, hippocampus, hypothalamic medial preoptic, paraventricular, and ventromedial nuclei were higher in the poly I:C group than those in the saline and heat-exposed groups on day 7, although the amount of interleukin-1 beta mRNA showed no differences. Serum adrenocorticotropic hormone and catecholamine levels were not significantly different between groups. The present results indicate that the prolonged fatigue induced by poly I:C, which is evaluated by the spontaneous running wheel activity, can be used as an animal model for the immunologically induced fatigue associated with viral infection, and suggest that brain IFN-alpha may play a role in this model.

Natural progression of untreated hepatolithiasis that shows no clinical signs at its initial presentation.

GOALS: To elucidate the natural progression of hepatolithiasis that showed no signs at the time of initial presentation. STUDY: Over a 17-year period, we observed 122 of 311 patients with hepatolithiasis who reported no symptoms and, thus, who received no treatment at initial presentation. The follow-up period was for up to 15 years (mean, 10.08 years). RESULTS: Fourteen of 112 patients (11.5%) developed some symptoms attributed to hepatolithiasis. The interval until the onset of symptoms ranged from 9 months to 7.33 years (mean, 3.42 years ). The developing symptoms included abdominal pain, hepatic abscess, cholangitis, and cholangiocarcinoma. Nine of the 14 patients (64.3%) developed stone migration to the extrahepatic bile duct at the onset of clinical symptoms. The incidence of lobar liver atrophy on computed tomography in the patients with symptomatic hepatolithiasis (13 of 14 patients; 92.9%) was significantly higher than that in the patients with asymptomatic hepatolithiasis (14 of 108 patients; 13.0%). The prognosis of the patients with symptomatic hepatolithiasis were as follows: 2 died of cholangiocarcinoma, 1 died of hepatic failure, and 11 survived. Fifteen of asymptomatic patients died, but none of these deaths were attributed to hepatolithiasis. CONCLUSIONS: Close observation is an alternative management at initial presentation for patients with asymptomatic hepatolithiasis without extrahepatic stones or lobar liver atrophy.

Long-term results of hepaticojejunostomy for hepatolithiasis.

The results of a hepaticojejunostomy as a biliary-enteric bypass for benign disease are usually excellent. On the other hand, hepatolithiasis features a high rate of residual and recurrent stones with cholangitis after surgery. This study aims to evaluate the long-term results of a hepaticojejunostomy for hepatolithiasis regarding both the degree of the occurrence of postoperative cholangitis and the outcome. The clinical records of 159 patients with hepatolithiasis who underwent surgical treatment over a 23-year period were also retrospectively reviewed. Ninety-four of 159 patients underwent a hepatecetomy and 65 patients were subjected to liver-preserving surgery by means of intra- and postoperative endoscopic lithotripsy. In addition 72 patients underwent a hepaticojejunostomy. The rate of residual or recurrent stones was 31.4 per cent after complete stone removal. Twenty-two (30.6%) of the 72 patients developed some kind of cholangitis. This rate was significantly higher than that (three of 87 patients) of the non-biliary-enteric anastomosis group regarding the occurrence of biliary complications. We conclude that the use of a hepaticojejunostomy for patients with possible residual stones or intrahepatic bile duct lesions remains controversial.

Apoptosis involved in density-dependent regulation of rat fibroblastic 3Y1 cell culture.

When rat 3Y1 fibroblastic cells are cultured toward confluency, the cells go through maximum cell density (overshoot) before reaching post-confluence stationary cell density. After overshooting, a number of floating cells are found in the medium. In a long-term culture, a cyclic change in cell number, an increase after each medium refreshment and subsequent cell loss within a few days has been observed so that the cell populations in the monolayer maintain post-confluence stationary cell density at a constant level. The floating cells excluded trypan blue, but they had no ability to attach to the substrate and to form colonies after being reseeded in fresh medium. They had condensed and uniformly electron-dense chromatin with sharply circumscribed edges. Their DNA contained a laddering pattern in harmony with internucleosomal cleavage. The features were those of apoptosis. When floating cells appeared, apoptotic bodies were also observed in the monolayer. Most of them were found within the cytoplasm of intact cells, suggesting that apoptotic bodies were also faded away from the culture by being rapidly engulfed by neighboring intact cells. These suggest that apoptosis and subsequent detachment from the monolayer or engulfment by neighboring intact cells, in addition to inhibition of cell division, are basic mechanisms on the process of density-dependent regulation in monolayer culture of rat 3Y1 cells.

[Selection of treatment in primary hepatolithiasis (PHL)].

Two hundred and three patients with PHL were encountered for the last 10 years. In order to select treatment of PHL, we classified PHL on the basis of atrophy of hepatic parenchyma (AHP), existence of extrahepatic gallstone and dilatation of extrahepatic bile duct (DEBD). For the case with AHP hepatic resection must be performed, because there is often chronic proliferating cholangitis and improvement in hepatic function can not be expected in view of AHP. For the case with extrahepatic gallstone without AHP hepatic resection. For the case with DEBD and without AHP extrahepatic gallstone must be removed. For the patient with extrahepatic gallstone and DEBD without AHP, indication of the removal of stones, depends on the existence of symptoms. Twenty-two cases treated by biliary drainage procedure often had some complications in postoperative course, that is 5 patients had cholangitis and 7 patients liver abscess. It was suggested, therefore, that biliary drainage procedure was contraindicated in PHL.

Isolation and identification of urinary nucleosides. Applications of high-performance liquid chromatographic methods to the synthesis of 5'-deoxyxanthosine and the simultaneous determination of 5,6-dihydrouridine and pseudouridine.

Modified nucleosides from pooled normal human urine were extracted using a boronate affinity gel column and fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC). The major constituents in each of the 30 RP-HPLC fractions were determined by gas chromatography-mass spectrometry of the trimethylsilyl derivatives of the fractions. The same RP-HPLC method was used in the synthesis of 5'-deoxyxanthosine from authentic 5'-deoxyadenosine. In addition, the simultaneous determination of urinary 5,6-dihydrouridine (D) and pseudouridine (psi) was carried out by RP-HPLC using two ODS columns in series. The level of D in pooled normal urine was 4.87 nmol/mumols creatinine. The RP-HPLC method was applied to the measurement of D and psi levels in urines collected before and after surgery from four patients with gastrointestinal cancer. A large decline in both nucleoside levels in urines after surgery was observed in three of the four cancer patients.

Analysis, identification and determination of urinary modified nucleosides of cancer and AIDS patients.

Modified nucleoside levels in urine samples collected before and after surgery from seven patients with malignant gastrointestinal cancer were examined by the reversed-phase HPLC method. Those of an AIDS patient, a breast cancer patient, and pooled normal urines were also compared. To monitor the effects of therapy on cancer patients, the levels of modified nucleosides, especially t6A and MTA, were found to be fairly effective. In an AIDS patient, the levels of most of the nucleosides exhibited remarkable increases compared to normal urine.

The identification of 5,6-dihydrouridine in normal human urine by combined gas chromatography/mass spectrometry.

The identification of 5,6-dihydrouridine in normal human urine is reported. Partial purification and isolation of the compound by boronate gel affinity chromatography and reversed-phase high-performance liquid chromatography preceded its characterization as a trimethylsilyl derivative by combined gas chromatography/mass spectrometry. Structure proof is based upon a comparison of mass spectral and chromatographic features of the urinary component to that of an authentic reference sample. Additional data derived from high resolution mass measurements and deuterium isotope-labeling experiments provide confirmation of fragment ion structure. The poor detectability inherent in the HPLC/uv analysis of nucleosides is also discussed.

Functional heterogeneity of human monocytes--with a special reference to flow cytometric assays.

A large number of human mononuclear cells were simultaneously separated into fractions enriched in B cells, T cells, large granular lymphocytes (LGL) and monocytes by centrifugal elutriation. Lymphocyte populations were analyzed using monoclonal antibodies. In particular, highly enriched natural killer cells, Leu7+ cells, were collected in the intermediate fractions. Monocytes, which were identified as esterase positive cells, and Leu M3 cells were collected at higher counterflow rates and in the final fraction. The purity of monocytes in the final fraction was 81%. The oxidative metabolic activity (H2O2 production) and non-specific esterase activity of individual monocytes was estimated in the analysis of functional heterogeneity of monocytes using flow cytometry. 2',7'-dichlorofluorescein diacetate (DCFH-DA) and fluorescein diacetate (FDA) were used as indicators in the measurement of H2O2 generation and esterase activity. Intracellular generation of a fluorescence product (H2O2 Production; average percentage of fluorescence positive cells) of monocytes in the stimulation of phorbol myristate acetate (PMA, 100 ng/ml) was greater in larger than smaller cells. H2O2 production gradually increased from 6% and 25-38% and 60% in the intermediate and final fractions respectively. Furthermore, the average fluorescence intensity of the large monocyte population in the final fraction was 1.13-1.31 fold more active than that of the smaller cells. Thus, the functional heterogeneity of human monocytes was further confirmed in the assays of H2O2 production exposed to PMA and FDA hydrolysis using flow cytometry. Furthermore, the CCE system can isolate lymphocyte subsets and LGL.

Mechanism of furosemide resistance in analbuminemic rats and hypoalbuminemic patients.

To elucidate the mechanism of resistance of hypoalbuminemic patients to furosemide, the effect of this diuretic on urine volume of normal and analbuminemic rats (NAR) and of hypoalbuminemic patients was studied. Intravenous administration of furosemide rapidly enhanced sodium diuresis in normal rats but not in NAR. Total plasma clearance and distribution volume of furosemide were much larger in NAR than in normal rats, while no significant difference in these pharmacokinetic parameters was observed for the unbound fraction of the diuretic between the two animal groups. In contrast, urinary secretion of furosemide was significantly lower in NAR than in normal rats. Injected furosemide bound to albumin markedly promoted diuresis in NAR, while the same dose of albumin alone had no effect, indicating that binding to albumin is essential for the delivery of furosemide to the kidney, the site for its action. Injection of the complex rapidly increased the urine volume of hypoalbuminemic patients who showed a marked resistance to this diuretic. Thus, the resistance to furosemide in both NAR and hypoalbuminemic patients may be explained on the same basis.

Further studies of lipid peroxidation in human paraquat poisoning.

In patients with subacute toxic reactions from paraquat poisoning (death within 11 to 41 days), the extent of lipid peroxidation, expressed as serum malondialdehyde level, was 2.7-fold higher (12.33 +/- 4.42 nmole/mL) before pulmonary fibrosis than that in normal controls (4.55 +/- 1.23 nmole/mL). The extent of lipid peroxidation in patients with acute toxic reactions (death within one to three days) was not elevated; these patients died of pulmonary edema and hemorrhage (acute respiratory distress), liver failure, renal failure, and adrenal necrosis. Remarkable high levels of paraquat (greater than 5 mg/L) were found in the urine, serum, and tissues of patients with acute toxic reactions; a small amount of paraquat was found in the serum or urine of patients with subacute toxic reactions five to 11 days after ingestion. Patients who survived had no elevation in lipid peroxidation. Administration of vitamin E (100 to 4,000 mg/day from the first hospital day) had no effect on survival.

Reversed-phase high-performance liquid chromatographic investigation of mucosal nucleosides and bases and urinary modified nucleosides of gastrointestinal cancer patients.

Reversed-phase high-performance liquid chromatography (HPLC) was used to determine the levels of nucleosides, bases and their metabolites in perchloric acid extracts of gastrointestinal mucosa. By comparing the levels of these compounds in the normal portion with the neoplastic portion of mucosa resected from malignant cancer patients, it was found that there was significant elevation of the uracil level in the neoplastic mucosa of all eight patients with colorectal cancer (2.7-fold in normal mucosa), but only in the neoplastic mucosa of one out of four patients with gastric cancer. The levels of hypoxanthine and uridine in the colorectal cancer mucosa samples and the inosine in gastric cancer samples were also significantly higher than those in normal mucosa. The urinary modified nucleosides were prefractionated with a boronate affinity gel column, and their levels were determined by the same HPLC method. There was no significant difference in the concentrations of pseudouridine, 1-methylguanosine N2-methylguanosine and N2,N2-dimethylguanosine between urine samples taken before and after surgery from eight patients with malignant colorectal cancer. Contrary to other reports, no significant differences in modified nucleoside levels were observed between urine samples from patients with colorectal cancer and those from normal subjects.

Reversed-phase liquid chromatographic investigation of nucleosides and bases in mucosa and modified nucleosides in urines from patients with gastrointestinal cancer.

Reversed-phase high-performance liquid chromatography (HPLC) has been used to determine the level of nucleic acid metabolites in perchloric acid extracts of gastrointestinal mucosa. By comparing the levels of these compounds in the normal portion with the levels in the neoplastic portion of mucosa resected from patients with malignant cancer, it was found that uracil was significantly elevated in the neoplastic colorectal mucosa (adenocarcinoma) of eight patients with colorectal cancer (P less than 0.01, statistically significant with the paired t test). The mean level of uracil in neoplastic colorectal mucosa was 2.7-fold higher than that in normal mucosa. However, in neoplastic gastric mucosa, only one out of four patients with gastric cancer showed elevated uracil. In neoplastic mucosa, the levels of hypoxanthine and uridine for colorectal cancer, and inosine for gastric cancer, were also significantly higher than those in normal mucosa (P less than 0.05, with the paired t test). The urinary modified nucleosides were prefractionated with a boronate affinity gel column, and their levels determined by the same HPLC method. No significant differences in the concentrations of pseudouridine, 1-methylguanosine, N2-methylguanosine or N2,N2-dimethylguanosine were observed in pre- and post-operative urines from patients with colorectal cancer and normal urines.

Acute renal failure in primary macroglobulinemia with small-molecule IgM.

Primary macroglobulinemia with acute renal failure developed in a 46-year-old man after administration of contrast medium (containing diatrizoate sodium and meglumine [Urografin]) for intravenous pyelography. On admission, the monoclonal protein peak could not be detected in serum by cellulose acetate electrophoresis, but the serial electrophoresis revealed a gradual increase in monoclonal protein peak, which later became two peaks. The serum of the patient contained the monoclonal small-molecule IgM kappa-chain protein with 13 to 14 Svedberg units in sedimentation constants, which is antigenetically the same as native IgM heavy chain. Although acute renal failure is extremely rare in primary macroglobulinemia, the small-molecule IgM seems to be one factor that induces acute renal failure.

Vibrio vulnificus septicemia.

A 33-year-old Japanese male, who had a three year history of biopsy-proved liver cirrhosis, was admitted to the hospital on June, 24, 1983 with a sudden onset of fever (38.6 degrees C), chills, generalized pain, nausea, anorexia, weakness, and eruption over the entire body. The patient went into shock and died about 7 hours after admission. Blood cultures before death were positive for V. vulnificus. Postmortem microscopic examination revealed "necrotizing vasculitis" in the small and large intestines, stomach, and skin, and also showed marked toxic epidermal necrolysis. This case matches the primary septicemia caused by V. vulnificus described by Blake et al. In addition, this case suggests that the septicemia was acquired through the gastrointestinal tract, especially the small intestine, because the V. vulnificus was isolated from blood and numerous Gram-negative bacilli around the submucosal vessels were observed in the area with acute necrotizing vasculitis.

Familial adult T-cell leukemia.

Two siblings who developed adult T-cell leukemia (ATL) are presented. The patient and 7 of 26 healthy family members examined had the serum antibodies against ATL-associated antigens (ATLA). This family study shows that two main routes of transmission of human T-cell leukemia virus (HTLV) may be involved: one is the route from parents to children and the other is horizontal transmission among spouses, especially from husband to wife; the anti-ATLA-positive family is considered to be a high-risk group for ATL.

Immunopathological characterization of the delayed hypersensitivity skin reaction to carrier protein.

Characteristics of the allergic skin reaction to free carrier protein in sensitized guinea pig with hapten-carrier conjugate have been studied. The animals sensitized with a low dose (4 micrograms) of dinitrophenylated bovine gamma globulin with Freund's complete adjuvant demonstrated a dermatitis when challenged intradermally with a small dose (10 micrograms) of bovine gamma globulin on the 10th or 14th day. The dermatitis was grossly and histologically similar to the classical form of delayed-type hypersensitivity skin reaction except lesser induration and less abundant neutrophil infiltration in the former. This reaction was clearly different from cutaneous basophil hypersensitivity because of the long lasting time course and fewer basophil infiltration in the former. No humoral antibody to carrier protein was detected in the sera of the sensitized animals by passive cutaneous anaphylaxis or by immunodiffusion in agar plates, and the state of the hypersensitivity could be passively transferred with peritoneal exudate cells and spleen cells. These observations indicated that the skin reaction to carrier protein might be of primarily delayed-type hypersensitivity. The vascular permeability change at the reaction sites was observed as a single phase delayed response and preceded the maximum erythema by about ten hours.