Development of a hybrid artificial liver using polyurethane foam/hepatocyte spheroid culture in a preclinical pig experiment.

We describe a preclinical study of our original hybrid artificial liver support system (HALSS) for a clinical trial. We designed a HALSS comprising a multi-capillary polyurethane foam packed-bed module (MC-PUF module) containing a total 200 g (2 x 10(10) cells) porcine hepatocytes, and an extracorporeal circulation device. Almost all porcine hepatocytes in the MC-PUF module formed many spherical multicellular aggregates (spheroids). This extracorporeal circulation device was improved to promote solute exchange between a living body and a MC-PUF module by including a plasma bypass line in the circulation loop. The efficacy of the HALSS was evaluated using a 25-kg pig with warm ischemic liver failure by portocaval shunt and ligation of hepatic artery (HALSS group, n=3). As a control experiment, the same system without hepatocytes in the module was used with the same kind of liver failure pig (Control group, n=3). The blood ammonia in the control group was 143 N-microg/dl at the start of circulation, and rapidly increased to 351 N-microg/dl at 2 hours and to 704 N-microg/dl at 6 hours. But the blood ammonia in the HALSS group was completely suppressed, and remained less than the hepatic coma level (over 200 N-microg/dl) during the circulation time. The blood glucose in the control group gradually decreased, and became less than 40 mg/dl within 6 hours of circulation. But the blood glucose in the HALSS group was maintained well, and remained the normal glucose level (50 - 105 mg/dl) for more than 20 hours of circulation. Improvement in blood creatinine and lactate, and the stabilization of vital signs and urinary excretion, were observed in the HALSS group. The survival time of the pigs in the HALSS group was 19.3 hours compared with 8.9 hours in the control group. In conclusion, our HALSS was effective to stabilize the general conditions of the body in addition to supporting various liver functions. These results suggest that our HALSS has a strong possibility to be used in treating liver failure patients. We have applied for approval of the clinical trial of our HALSS to our institutional ethics committee.

Recovery of rats with fulminant hepatic failure by using a hybrid artificial liver support system with polyurethane foam/rat hepatocyte spheroids.

We studied the recovery of rats with fulminant hepatic failure (FHF) by treating them with our original hybrid artificial liver support system (HALSS). FHF was induced by a two-thirds partial hepatectomy and 10 minutes of hepatic ischemia. Rats with FHF were treated with a polyurethane foam/spheroid HALSS including 2.0 x 10(8) hepatocytes for 1 hour (HALSS group, n = 5), and with the same system without hepatocytes in the artificial liver module as a control experiment (sham-HALSS group, n = 3). The level of blood constituents, ammonia, glucose and creatinine, showed no major difference between the two groups at the end of treatment. All rats in the sham-HALSS group died within 5 hours after treatment. However, the level of blood constituents of rats with FHF in the HALSS group improved with time, and all rats in the HALSS group recovered. Liver tissue of rats treated with HALSS showed cell mitosis and improvement from injury. These results indicated that our HALSS has a strong possibility to induce recovery from hepatic failure.

The efficacy of nafamostat mesilate on the performance of a hybrid-artificial liver using a polyurethane foam/porcine hepatocyte spheroid culture system in human plasma.

Nafamostat mesilate (FUT) is a protease inhibitor of complement activation. The present study investigates whether FUT protects porcine hepatocytes from being injured by human plasma in a multi-capillary polyurethane foam packed-bed culture system (MC-PUF) such as the hybrid-artificial liver (PUF-HAL). Human plasmas with 1 mM of added ammonia were perfused using a small-scale PUF-HAL with porcine hepatocytes. FUT was continuously infused (10 microg/ml, 50 microg/ml). The ammonia detoxification was maintained in human plasma for 24 hours and for 48 hours with FUT which suppressed the rapid increase of asparaginic acid aminotransferase (AST) and alanine aminotransferase (ALT). After 60 hours of perfusion, hepatocyte spheroids completely collapsed in the human plasma, but a small amount of hepatocyte spheroid was maintained by FUT. The effect of FUT was slightly greater at 50 microg/ml than at 10 microg/ml. Our results suggest that FUT has protective effects against porcine hepatocytes in human plasma, and our PUF-HAL using porcine hepatocytes can function in human plasma for about 48 hours with FUT.

Polyurethane foam/spheroid culture system using human hepatoblastoma cell line (Hep G2) as a possible new hybrid artificial liver.

The risk of xenozoonosis infections poses the greatest obstacle against the clinical application of hybrid artificial liver support system (HALSS). Primary human hepatocytes are an ideal source for HALSS, but the shortage of human livers available for hepatocyte isolation limits this modality. To resolve this issue, we used human hepatocytes with replication capacity (fetal hepatocytes, Hep G2, and Huh 7) in a polyurethane foam (PUF)/spheroid culture system in vitro, and analyzed liver functions such as ammonia removal and albumin synthesis capacity; results were compared to those of porcine hepatocytes. Human fetal hepatocytes, Hep G2, and Huh 7 formed spheroids spontaneously within 24 h in a PUF/spheroid culture system; ammonia removal activity (micromol/10(6) nuclei/h) was upregulated, as was albumin synthesis activity (microg/10(6) nuclei/day). In particular, Hep G2 spheroids demonstrated high ammonia removal and albumin synthesis activities: 85% of the ammonia removal activity and 171.7% of the albumin synthesis activity of porcine hepatocytes in the monolayer culture. These results indicate the possibility of the development of a multicapillary PUF (MC-PUF) packed-bed culture system of hepatocyte spheroids as a HALSS using Hep G2.

Mass preparation of primary porcine hepatocytes and the design of a hybrid artificial liver module using spheroid culture for a clinical trial.

To isolate a large number of porcine hepatocytes, we originally developed a mass preparation method that combined the usual collagenase perfusion method of a whole liver with a collagenase redigestion method of tissue fragments after liver perfusion. Using a pig of 10kg, collagenase perfusion only resulted in a yield of 63+/-78 x 10(8) total cells with a viability of 69.2+/-25.3 %, but our combined method had a yield of 167+/-31 x 10(8) total cells with a viability of 87.9+/-4.4% (mean +/- SD). Also, the combined method was applied to two pigs of 10kg body weight at the same time, and isolated 387+/-89 x 10(8) hepatocytes with a viability of 87.1+/-6.9% and a purity of 93.6+/-2.8 % in 11 experiments. We designed a large multi-capillary polyurethane foam (MC-PUF) packed-bed module containing 1 x 10(10) porcine hepatocytes on a clinical trial scale. The porcine hepatocytes in the module formed spherical multicellular aggregates (spheroids) of 200 - 500 microm diameter. Most hepatocytes forming spheroids were viable judged by fluorescein diacetate and ethidium bromide staining. The activities of ammonia removal, albumin secretion and oxygen consumption of the large MC-PUF module were the same as for a small MC-PUF module containing 2 x 10(8) porcine hepatocytes, and were maintained for at least 9 days of culture. These results show that a large MC-PUF module is successfully scaled up 50 times. In conclusion, we succeeded in developing a mass preparation method of porcine hepatocytes and a large hybrid artificial liver module on a clinical trial scale.

Approach to patients with hirsutism.

Hirsutism is a common medical condition that in most women is due to the polycystic ovary syndrome or is idiopathic. For a few women, hirsutism signals a serious underlying disorder such as an ovarian or adrenal tumor, congenital adrenal hyperplasia, or Cushing's syndrome. A detailed medical history and examination can identify women in whom a serious disease is suspected and for whom laboratory evaluation is warranted. Measurements of serum testosterone, dehydroepiandrosterone, and 17 alpha-hydroxyprogesterone levels, and 24-hour urinary cortisol concentrations are important screening tests. Therapy is directed at suppressing ovarian or adrenal androgen production, inhibiting the conversion of testosterone to dihydrotestosterone, or antagonizing the effects of androgens at the receptor level.

Thyroiditis: a clinical review.

Thyroiditis is a spectrum of diseases that includes acute, subacute and chronic thyroiditis, all of which demonstrate the histologic findings of inflammation, fibrosis or lymphocytic infiltration. Acute thyroiditis is an infectious disease characterized by the abrupt onset of anterior neck pain and swelling. Subacute thyroiditis can have a viral or autoimmune etiology and may present with or without thyroid pain. Chronic thyroiditis is one of the most common causes of asymptomatic goiter and hypothyroidism. Each type of thyroiditis has a distinct clinical presentation and laboratory profile that allows the clinician to identify the disease and provide proper management.

Influx of testosterone-binding globulin (TeBG) and TeBG-bound sex steroid hormones into rat testis and prostate.

The availability of testosterone and estradiol to Sertoli and prostate cells is dependent upon 1) the permeability properties of the blood-tubular barrier (BTB) of the testis or prostate cell membrane, and 2) sex steroid binding to plasma proteins, such as albumin or testosterone-binding globulin (TeBG). Sex steroid influx into these tissues was studied after in vivo arterial bolus injections of [3H]testosterone or [3H]estradiol in anesthetized rats. Both testosterone and estradiol were readily cleared across the BTB or prostate cell membrane in the absence of plasma proteins and in the presence of human pregnancy serum, in which testosterone or estradiol are 80-95% distributed to TeBG. The extravascular extraction of [3H]TeBG across the BTB or prostate plasma membrane [73 +/- 2% (+/- SE) and 92 +/- 9%, respectively] was significantly greater than extraction of [3H]albumin or other plasma space markers and indicative of a rapid first pass clearance of TeBG by Sertoli or prostate cells. In summary, these studies indicate that 1) testosterone and estradiol are readily cleared by Sertoli and prostate cells; 2) albumin- and TeBG-bound sex steroids represent the major circulating pool of bioavailable hormone for testis or prostate; and 3) the TeBG-sex steroid complex may be nearly completely available for influx through the BTB or prostate plasma membrane.

Silent thyroiditis.

Silent thyroiditis is an increasingly recognized cause of transient thyrotoxicosis. Inflammatory destruction of thyroid follicles results in release of preformed thyroxine and triiodothyronine. Patients present with symptoms of thyrotoxicosis, but unlike subacute thyroiditis, lack thyroid pain or tenderness. The thyrotoxic state spontaneously resolves in 2 to 12 weeks at which time the patient either returns to a euthyroid state or passes through a transient hypothyroid phase. Diagnostic laboratory findings include elevations of thyroxine and triiodothyronine and a markedly depressed radioactive iodine uptake. It is imperative for the primary care physician to distinguish silent thyroiditis from chronic causes of hyperthyroidism, eg, Graves' disease, since treatment must be palliative rather than definitive. Long-term prognosis is usually excellent.

Restricted transport of vitamin D and A derivatives through the rat blood-brain barrier.

The present studies measure the transport of retinol, retinoic acid, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], and 25-hydroxyvitamin D3 [25-(OH)D3] through the rat brain capillary endothelial wall, i.e., the blood-brain barrier (BBB). The vitamin A and D derivatives bind both to albumin and to specific high-affinity binding proteins in plasma. In the presence of physiologic concentrations of plasma proteins, the extraction by brain of all four compounds was 5% or less.

Cyclic Cushing's syndrome.

Cyclic Cushing's syndrome is a rare but increasingly recognized disorder of periodic fluctuations of adrenal steroid production. A case of cyclic Cushing's syndrome due to a pituitary adenoma is described. The patient demonstrated a prolonged cycle length of approximately six months, during which a spontaneous remission occurred both clinically and biochemically. Previously documented cases of cyclic Cushing's syndrome are reviewed, and the pitfalls in interpretation of results of dexamethasone suppression testing in the presence of spontaneous fluctuations in cortisol production are discussed.

Blood-brain barrier transport and brain sequestration of propranolol and lidocaine.

Lipophilic amine drugs such as propranolol and lidocaine are actively sequestered by tissues via saturable cytoplasmic binding systems. The present studies were designed to characterize the kinetics of drug transport and sequestration in rat brain in vivo by using the carotid injection technique. Both propranolol and lidocaine are sequestered by brain, and the half time (t 1/2) of clearance of the drugs from brain to blood is 6-7 min. The t 1/2 of propranolol association and dissociation reactions with the brain sequestration system are 0.38 +/- 0.03 and 1.33 +/- 0.20 min, respectively. The blood-brain barrier transport of propranolol and lidocaine is inhibited by acid pH, and drug transport is mediated by a low-affinity, high-capacity saturable transport system [propranolol half-saturation constant (Km) = 9.8 +/- 1.2 mM, maximal rate of saturable transport (Vmax) = 5.7 +/- 0.7 mumol X min-1 X g-1, and nonsaturable transport constant (KD) = 0.061 +/- 0.012 ml X min-1 X g-1). These studies indicate that in addition to cerebral blood flow, the distribution of lipophilic amines in brain is a function of plasma pH and of the activity of brain sequestration systems. The latter may represent high-capacity cytoplasmic drug-binding proteins that normally bind endogenous ligands in brain.

Human insulin.

Human insulin may be advantageous for certain subsets of patients, such as those with gestational diabetes and those who need insulin only during stress or surgery. To date, there is no evidence to support the use of human insulin in diabetics who are doing well on older insulin preparations.

Galactorrhea and hyperprolactinemia.

PIP: Galactorrhea refers to the secretion of a milky fluid from the breast, occurring either spontaneously or with manual expression, in the absence of pregnancy or the postpartum state. The development of a sensitive and specific radioimmunoassay for human prolactin during the last decade along with improved neuroradiologic techniques have greatly expanded understanding of nonpuerperal lactation. Discussion reviews the pevalence of galactorrhea, the etiology of hyperprolactinemia and/or galactorrhea, pituitary tumor, parapituitary lesions, oral contraceptive (OC) associated galactorrhea, drug induced galactorrhea, hypothyroidism, neurogenic stimulation, idiopathic galactorrhea, clinical features of galactorrhea, laboratory evaluation, neuroradiologic evaluation, hypocycloidal tomography, carotid angiography, pneumoencelphalography (PEG), computerized tomography (CT), management, and therapy. The prevalence of galactorrhea in women is reported to range from 0.1-32%. Much of this variability can be attributed to differences in examination techniques, the investigator's definition of galactorrhea, and the patient population studied. Nonpuerperal hyperprolactinemia is an important cause of galactorrhea and is found in 49-77% of cases, but hyperprolactinemia does not appear to be the sole prerequisite for galactorrhea since only 15-68% of patients with excessive prolactin secretion develop galactorrhea. Pituitary tumors are the most important diagnostic consideration have been reported in approximately 20% of cases of galactorrhea and 34% of cases of amenorrhea galactorrhea. Parapituitary lesions, an infrequent but important cause of hyperprolactinema and galactorrhea, can result in excessive prolactin secretion by interfering with either the production or delivery of prolactin inhibitory factor to the lactotrope cells of the anterior pituitary gland. OC associated galactorrhea is a frequent diagnosis in patients with nonpuerperal lactation and has been implicated in 10-14% of cases. OC discontinuation can also produce galactorrhea. Pharmacologic agents are a common cause of hyperprolactinemia and/or galactorrhea. A variety of forms of neurogenic stimulation have been linked to hyperprolactinemia and/or galactorrhea. Idiopathic galactorrhea is a diagnosis of exclusion and is applicable to 40-50% of patients with nonpuerperal lactation. In patients with galactorrhea, the presence of the abnormal, milky breast secretion may be the sole symptom or it may be associated with other pertinent clinical findings. All patients with galactorrhea should have measurements of thyroid stimulating hormone and serum prolactin performed. The optimal management of patients with prolactinomas is somewhat controversial. Surgical resection remains the most definitive form of therapy with the transsphenoidal approach most frequently used. Recent invesitgations show promise for bromocriptine as an alternative therapeutic modality.^ieng

Protein-bound corticosteroid in human serum is selectively transported into rat brain and liver in vivo.

It is generally regarded that only free corticosteroid is available for entry into tissues in vivo, although some studies have suggested that albumin-bound corticosteroid is available for liver uptake. However, recent studies suggest that owing to favorable kinetic relationships among tissue capillary transit times and hormone dissociation rates from plasma proteins, free plus albumin-bound hormone may be available to peripheral tissues. Moreover, globulin-bound hormone may enter the liver under normal conditions and be available to peripheral tissues under pathological circumstances. The present studies measure the free and noncorticosteroid-binding globulin (non-CBG)-bound corticosteroid fractions in vitro and compare these measurements to the fraction of corticosteroid in human sera that is available for entry into rat brain and rat liver in vivo. Corticosterone was used as the model corticosteroid, since this compound binds to CBG with the same affinity as does cortisol, and the brain extraction of corticosterone is more readily measured in vivo than is the brain extraction of cortisol. Serum was obtained from 51 human subjects and included samples from newborns, pregnant mothers, normal subjects, and patients with either cirrhosis or renal failure. Serum levels of CBG varied more than 6-fold, and both the free and the non-CBG-bound fractions were generally inversely related to the CBG concentration. The fraction of corticosterone available for entry into the brain was much greater than the free fraction, but was not significantly different from the non-CBG-bound moiety. Moreover, the rate of corticosterone dissociation from CBG (t 1/2 = 27 +/- 1 sec at 22 C) was not increased in any of the serum samples studied. The fraction of corticosterone available for uptake by the liver was up to 3-fold greater than that of the non-CBG fraction and had no relationship with the serum concentration of CBG. These studies indicate that albumin-bound, but not globulin-bound, corticosteroid is available for entry into a peripheral tissue such as the brain. However, globulin-bound corticosteroid is readily transported into the liver. It is suggested that the routine measurements of the non-CBG-bound corticosteroid provide a more accurate index of the corticosteroid available to peripheral tissues in vivo than does the measurement of free corticosteroid.

Transport of propranolol and lidocaine through the rat blood-brain barrier. Primary role of globulin-bound drug.

Basic lipophilic drugs such as propranolol and lidocaine are strongly bound by alpha(1)-acid glycoprotein, also called orosomucoid. Although the liver is known to rapidly clear plasma protein-bound propranolol or lidocaine, it is generally regarded that peripheral tissues, such as brain or heart, are only exposed to the small fraction of drug that is free or dialyzable in vitro. The "free drug" hypothesis is subjected to direct empiric testing in the present studies using human sera and an in vivo rat brain paradigm. Serum from 27 human subjects (normal individuals, newborns, or patients with either metastatic cancer or rheumatoid arthritis) were found to have up to a sevenfold variation in orosomucoid concentrations. The free propranolol or lidocaine as determined in vitro by equilibrium dialysis at 37 degrees C varied inversely with the orosomucoid concentration. Similarly the rate of transport of propranolol or lidocaine through the blood-brain barrier (BBB) was inversely related to the existing serum concentration of orosomucoid. However, the inhibition of rat brain extraction of drug by orosomucoid in vivo was only about one-fifth of that predicted by free drug measurements in vitro. This large discrepancy suggested orosomucoid-bound drug was readily available for transport into brain in vivo. Studies using purified human orosomucoid in the rat brain extraction assay also showed that orosomucoidbound propranolol or lidocaine is readily transported through the BBB. Conversely, albumin-bound propranolol or lidocaine was not transported through the BBB. The studies using albumin provide evidence that the in vivo rat brain paradigm used in the present investigations is capable of confirming, when possible, predictions made by the "free drug" hypothesis. These data suggest that the amount of circulating propranolol or lidocaine that is available for transport into a peripheral tissue such as brain is not restricted to the free (dialyzable) moiety but includes the much larger globulin-bound fraction. Therefore, existing pharmacokinetic models should be expanded to account for the transport of protein-bound drugs into peripheral tissues similar to what is known to occur in liver.

Effects of human cirrhotic serum on estradiol and testosterone transport into rat brain.

It is known that sex hormone-binding globulin (SHBG), which binds both testosterone and estradiol in human serum, is markedly elevated in cirrhosis. In addition, it is known that the net metabolic clearance of testosterone is decreased in cirrhosis, while no change in the metabolic clearance of estradiol is observed. Since net clearance estimates are not direct measures of plasma protein binding effects, the present studies were designed to examine the effects of cirrhotic human serum on the unidirectional clearance of sex steroids using an in vivo rat brain paradigm previously described. Cirrhotic serum was characterized by the following changes from control serum: 2.6-fold increase in SHBG, 40% decrease in albumin, and 30% and 22% decreases in the dialyzable fractions of testosterone and estradiol, respectively. In addition, the non-SHBG-bound fraction of estradiol was decreased 41% in cirrhosis, but no significant change in the brain extraction of estradiol was observed using cirrhotic serum. In contrast, the unidirectional testosterone clearance was decreased 33% by cirrhotic serum. These studies indicate that changes in the net metabolic clearance of sex steroids closely parallel the changes in unidirectional clearance caused by alterations in plasma proteins. The absence of a decrease in estradiol clearance in cirrhosis in association with the substantial decrease in the non-SHBG-bound estradiol fraction is an unexpected finding, since previous studies have shown that a close parallel exists between these two parameters. A possible explanation is that estradiol bound to the SHBG in human cirrhotic serum is partially available for transport into peripheral tissues such as the brain.

Posttraumatic epilepsy and CT scan.

Among 83 head-trauma cases examined by CT scan in a later year, 41 were included in a seizure group of those who clinically showed late epilepsy and who obviously showed epileptic discharge such as spike or spike and wave in EEG after trauma, and 42 were included in a nonseizure group of those who had some sequelae such as abnormal findings in EEG or neurologic defects: the CT findings of these 83 cases were collected and compared with clinical findings, EEG, and other data. The CT findings of these cases were divided into five groups: (A) a normal group; (B) a group showing partial or unilateral ventricular dilatation; (C) a group showing porencephaly in the cerebral parenchyma; (D) a group suspected of cortical atrophy; (E) a group of other cases. Group D contained 44.3% of the cases, and was the largest group; the frequency of seizure was highest in Group C. The CT classification reveals the contradiction of the conventional definition of posttraumatic epilepsy and also poses some problems concerning the disease. CT scan is mostly applied to head trauma in the acute stage, and as for trauma in the chronic stage there are a few reports on chronic subdural hematoma and post-traumatic hydrocephalus. We noticed posttraumatic epilepsy among the sequelae of trauma, and then analyzed CT findings of the epilepsy: the results are reported here.