Relationship between histopathologic typing and morphonuclear assessments of 238 thyroid lesions. Digital cell image analysis performed on Feulgen-stained nuclei from formalin-fixed, paraffin-embedded materials.

A digital cell image processor was used to compute 15 parameters on Feulgen-stained thyroid nuclei from 238 archival, i.e., formalin-fixed, paraffin-embedded thyroid lesions. The morphonuclear parameters were related to morphometric (nuclear area), densitometric (nuclear DNA content), and textural (chromatin pattern characteristics) features. With respect to development of a malignant condition, their variations were compared with the World Health Organization's International Histological Classification of thyroid tumors. The relationship between morphonuclear parameter assessments and tumor size and the presence or absence of metastasis at the time of diagnosis was also investigated: no relationship with respect to cytomorphonuclear assessments was found. Nuclear area and nuclear DNA content discriminated between simple multinodular goiters and multinodular goiters with adenomatous hyperplasia. In the same way, the mean parameter values describing the chromatin pattern of multinodular goiters were significantly distinct from those describing the chromatin pattern of adenomas and carcinomas. Furthermore, chromatin pattern descriptions made it possible to discriminate between cell nuclei from papillary carcinomas and follicular carcinomas, and further between follicular carcinomas and follicular adenomas. A marked variation was observed within individual cases of multinodular goiters, adenomas, and carcinomas, a feature suggesting that morphonuclear assessment is of limited value for the diagnosis of individual clinical cases. In contrast, these results show that morphometric, densitometric, and textural nuclear assessments are useful aids for thyroid tumor typing, adding interesting results with respect to thyroid tumor progression. Indeed, the primary findings demonstrate that nodules with adenomatous hyperplasia from multinodular goiter lesions and microvesicular adenomas more closely resemble follicular carcinomas than do simple multinodular goiters and normomacrovesicular adenomas. Such data might reflect a biologic progression from benign to malignant follicular thyroid lesions.


Assessment of different methods to detect increased autochthonous production of immunoglobulin G and oligoclonal immunoglobulins in multiple sclerosis.

The formulae for immunoglobulin G (IgG) synthesis rate, IgG(loc), IgG index, and IgG extended index in determining increased autochthonous production (intra-blood-brain barrier synthesis) of IgG was assessed in 50 chronic progressive multiple sclerosis patients. The results of all four formulae clearly show elevated intra-blood-brain barrier IgG synthesis in 35 patients, 94% of whom had oligoclonal immunoglobulins detected by the method of isoelectric focusing with immunofixation and silver staining (IEF-IS). The results from the four formulae are all normal in seven patients (none have oligoclonal immunoglobulins by IEF-IS) and are discordant in eight patients (five have oligoclonal immunoglobulins by IEF-IS). Whenever intra-blood-brain barrier synthesis of IgG is suspected, IEF-IS should be used to assay for oligoclonal immunoglobulins because this method is more sensitive than agarose electrophoresis with either Coomassie blue dye or silver stain (94% vs. 74% vs. 54%).


Sexual abuse of children. The detection of semen on skin.

The detection of semen on the skin of children who present within 72 hours of an episode of sexual assault is critical to medical, forensic, and legal personnel. The Wood's Lamp, a UV light that causes semen to fluoresce, and four forensic laboratory techniques were compared to determine their sensitivity and decline in sensitivity over time. A descriptive study. Eleven adult female volunteers. Semen was placed on the skin of the volunteers. Samples of the dried semen were assessed during a 28-hour period with the Wood's Lamp, microscopy, the acid phosphatase assay, and two assays for the prostatic protein p30 (counterimmunoelectrophoresis and enzyme-linked immunosorbent assay). The intensity of the Wood's Lamp fluorescence of semen diminished dramatically by 28 hours; in contrast, the fluorescence of urine persisted up to 80 hours. Over time, the p30-enzyme-linked immunosorbent assay technique was more sensitive than microscopy, the acid phosphatase assay, and p30-counterimmunoelectrophoresis in detecting semen on skin. The Wood's Lamp is not a sensitive screening tool and should be used with caution. To improve the detection of sexual abuse in children, we recommend that the p30-enzyme-linked immunosorbent assay be used because of its potential as a more sensitive assay than those in current clinical use.


Risk factors for surgery for prostatic hypertrophy.

The purpose of this case-control study was to evaluate potential risk factors for prostatic hypertrophy. The cases were 910 residents of Rhode Island who had a partial or total prostatectomy that was not related to cancer in the years 1985-1987. The controls were 2,003 members of the source population who were selected from a list of holders of Rhode Island driver's licenses or a roster of older Americans compiled by the Health Care Financing Administration. Cases and controls were interviewed by telephone. The risk of prostatic hypertrophy was elevated in Jewish men compared with Protestants and Catholics and in blacks compared with whites. Risk was reduced in ever-married compared with never-married men, in men who had left school at age 16 years or more compared with those who had left earlier, and in relatively tall or relatively heavy men. Coffee drinking and cigarette smoking were inversely but only weakly related to prostatic hypertrophy. There was a relatively strong, although irregular, inverse relation of beer drinking to prostatic hypertrophy. The associations of spirits and wine consumption with prostatic hypertrophy were weak.


Home visitation for pregnant women and parents of young children.

Many of the most pervasive, intractable, and costly problems faced by high-risk women and young children in our society today are a consequence of adverse maternal health-related behaviors (such as cigarette smoking, drinking, and drug use during pregnancy), dysfunctional infant care giving, and stressful environmental conditions that interfere with individual and family functioning. These problems include low birth weight, child abuse and neglect, childhood injuries, unintended and closely spaced pregnancy, and reduced economic self-sufficiency on the part of parents. Evidence is accumulating that these problems can be reduced with comprehensive programs of prenatal and infancy home visitation by nurses. While we are witnessing a renaissance of interest in home visitation as a means of addressing these problems, the recommendations of various health and human service advisory groups about the structure of proposed home-visitation initiatives are uncoordinated and frequently inconsistent with the empirical evidence. Home visitation is a promising strategy, but only when the program meets certain standards. The more successful programs contain the following: (1) a focus on families at greater need for the service, (2) the use of nurses who begin during pregnancy and follow the family at least through the second year of the child's life, (3) the promotion of positive health-related behaviors and qualities of infant care giving, and (4) provisions to reduce family stress by improving the social and physical environments in which families live.


Nonlissencephalic cortical dysplasias: correlation of imaging findings with clinical deficits.

To establish correlations between MR patterns and clinical outcome in patients with nonlissencephalic cortical dysplasias. MR and clinical data from 36 patients with cerebral cortical gyral anomalies (other than classical type I or type II lissencephaly) were retrospectively reviewed. The five patients with diffuse cortical dysplasia, including two with congenital infections, had microcephaly and severe development delay from a very early age. Infantile spasms occurred in three of the five. Focal areas of cortical dysplasia were most common in the frontal lobes, but were seen in all areas of the brain. The most common MR appearances were 1) a thickened, irregularly bumpy cortex with shallow, wide sulci, and 2) a deep infolding of thickened cortex. The twelve patients with bilateral focal dysplasia had a high incidence of bilateral motor dysfunction (67%), delayed speech (67%), and generalized developmental delay (92%). When the dysplasia was unilateral, contralateral spastic hemiplegia or monoplegia was present in 14 of 19 patients (74%), but dysphasia was uncommon, even in patients with dysplasia in the frontal lobe of the dominant hemisphere. Surgical resection of focal areas of cortical dysplasia in patients with medically refractory seizures is becoming more common, and the neuroradiologist will play an increasingly important role in the initial diagnosis and delineation of these anomalies.


Alpha-fetoprotein levels in normal adults.

Alpha-fetoprotein (AFP) is a fetal specific glycoprotein normally produced primarily by the fetal liver. Normally, AFP levels decline rapidly after birth, reaching undetectable levels (less than 10 ng/ml) within several months after birth. The authors have developed a more sensitive radioimmunoassay, which has allowed them to study low levels of AFP in normal adults and to determine factors which may affect its normal level. Two hundred and seventy normal Houston blood donors were screened for the absence of hepatitis B and normal ALT levels. The mean AFP level was 3.04 ng/ml +/- 1.9 SD. There was a statistically significant higher level in men compared to women (p less than .004). Regression analysis demonstrated a statistically significant increase of AFP levels with age both in men (p less than .05) and in women (p less than .01). These data delineate the normal level of serum AFP in normal adults in the United States. With the normal level now defined, it becomes possible to compare levels in different populations including those exposed to hepatotoxins or hepatocarcinogens in the environment.


The California Maternal Serum alpha-Fetoprotein Screening Program: the role of ultrasonography in the detection of spina bifida.

Between January 1988 and June 1990, 161 cases of open spina bifida were identified by the California Maternal Serum alpha-Fetoprotein Screening Program. Eight percent of these cases were not diagnosed by an initial ultrasonographic evaluation. Three defects were not recognized until birth. Ultrasonography is inadequate to identify all cases of open spina bifida.


Maternal serum alpha-fetoprotein, beta-human chorionic gonadotropin, and unconjugated estriol levels in midtrimester trisomy 18 pregnancies.

The purpose was to evaluate the levels of maternal serum human chorionic gonadotropin, alpha-fetoprotein, and unconjugated estriol in trisomy 18 pregnancies compared with normal singleton pregnancies. Sera from 14 trisomy 18 pregnancies (13 retrospectively and one prospectively ascertained) were analyzed for human chorionic gonadotropin, alpha-fetoprotein, and unconjugated estriol. The alpha-fetoprotein levels in the 10 trisomy 18 pregnancies without open neural tube or ventral wall defect had a median of 0.65 multiple of the median, although two had alpha-fetoprotein levels above 2.5 multiples of the median. The human chorionic gonadotropin levels had a median of 0.32 multiple of the median and the unconjugated estriol levels had a median of 0.56 multiple of the median. Although most women with trisomy 18 pregnancies had serum human chorionic gonadotropin levels that were less than 1.0 multiple of the median, three had markedly elevated human chorionic gonadotropin levels (greater than 5.0 multiples of the median). Our data are partially consistent with those previously published but suggest the possibility of a bimodal distribution of alpha-fetoprotein and human chorionic gonadotropin levels in trisomy 18-affected pregnancies, unrelated to a neural tube or abdominal wall defect. The efficiency of screening for trisomy 18 prospectively, using the three serum markers, requires further evaluation.


Chorionic villus sampling for fetal Rh typing: clinical implications.

The prenatal Rh typing of red blood cells obtained by chorionic villus sampling was performed with an immune rosette technique. Ten Rh-negative pregnant women undergoing chorionic villus sampling at 9 to 11 weeks' gestation were studied at a large referral hospital. Accurate Rh phenotyping may be done on red blood cells obtained from chorionic villi weighing 2 to 8 mg. The preparations were shown to be greater than 90% fetal in origin as demonstrated by radial immunodiffusion quantitation of fetal hemoglobin-containing cells. Of the 10 fetuses typed in the first trimester nine of the pregnancies were carried to term. In all cases typing of red blood cells confirmed the antenatal fetal red cell typing. This study shows that antenatal Rh phenotyping may be performed as early as 9 to 11 weeks' gestation. This technique may be used in the management of pregnancies complicated by Rh incompatibility.


Surgery for disseminated abdominal sarcoma.

Seventy-two consecutive patients with disseminated soft tissue sarcoma in the abdomen were prospectively placed in a program of debulking surgery. The tumor was completely resectable in 64% of the patients. Following the first exploration, the median survival was 23 months for those with resection of metastases and 9 months for those without resection (p less than or equal to 0.01); the former group had a survival rate of 28% at 3 years, 18% at 4 years, and 4% at 5 years (44%, 37%, and 10%, respectively, for low-grade sarcomas, i.e., grade I or II sarcomas), whereas in the latter group, none survived for 3 years. In the group with resection, patients with grade III tumors had a median survival longer by 6 months, and those with low-grade tumors by 28 months (p less than or equal to 0.001), over the respective median survival of patients with unresectable tumors. Metastasectomy appeared to prolong survival in all patients and significantly so in patients with low-grade tumors and those with long disease-free intervals.


Detection of proteins and phenol in DNA samples with second-derivative absorption spectroscopy.

We have employed near-uv second-derivative spectra of DNA, N-acetyl-L-tryptophanamide, N-acetyl-L-tyrosinamide, N-acetyl-L-phenylalanine ethyl ester, and phenol in a matrix least-squares multicomponent analysis algorithm to detect the presence of tryptophan, tyrosine, phenylalanine, and/or phenol in DNA preparations. With this method, each of these compounds can be detected in a DNA sample (absorbance, 0.1) at absorbance levels of less than 0.002. In practice, the presence of proteins can be detected at absorbance levels of less than 0.003. Using second-derivative spectra of proteins, contents of mixtures of proteins and DNA can be determined with less than 1% error. Mixtures of DNA and RNA can also be quantitatively analyzed with an error of approximately 2%. This technique can be easily implemented with computer-controlled spectrophotometers equipped with standard spectral analysis software. With prerecorded standard spectra, the time of analysis does not exceed a few seconds.


Will the calcium channel agonist BAY K8644 inhibit halothane-induced impairment of calcium current?

The inhaled anesthetics impair transsarcolemmal calcium entry (ICa) in myocardial cells, although the mechanism of this interaction is not known. This inhibition of calcium entry has been implicated in the myocardial depression of the volatile anesthetics. To further characterize this interaction and to evaluate whether a calcium channel agonist could attenuate or prevent the inhibition of calcium entry, the effect of the calcium channel agonist BAY K8644 on the impairment of ICa by halothane was evaluated in single guinea pig ventricular myocytes. Calcium currents were evoked by means of the whole-cell voltage-clamp technique. Baseline peak ICa was higher in the cells exposed to 5 microM BAY K8644 (311 vs 206 pA/cm2, P less than 0.04). On exposure to 1% halothane, peak ICa was impaired to an identical degree whether or not cells were exposed to BAY K8644 (78% and 79% of baseline value). This is consistent with the suggestion that the effects of these agents on ICa are nonspecific. However, the increase in ICa suggests that appropriate calcium channel agonists might serve to ameliorate the myocardial depressant effects of halothane.


Calcium channels are involved in the hypnotic-anesthetic action of dexmedetomidine in rats.

Our study examined whether calcium channels are involved in the anesthetic action of dexmedetomidine (100-300 micrograms/kg), a highly selective alpha 2-adrenoceptor agonist. To investigate this, we studied the effects of verapamil (1.25 or 2.5 mg/kg), a calcium channel blocker, and BAY K8644 (0.5 or 1 mg/kg), a calcium channel agonist, on the hypnotic-anesthetic effect of dexmedetomidine in rats. Loss of the righting reflex was used to determine the presence of anesthesia, and its length in minutes was referred to as the duration of hypnosis. Verapamil significantly enhanced the duration of the hypnotic-anesthetic action of dexmedetomidine (P less than 0.05). In contrast, BAY K8644 caused a significantly increased onset of hypnosis (P less than 0.001) and attenuated the anesthetic property of dexmedetomidine. These results suggest that the hypnotic-anesthetic action of dexmedetomidine is influenced by the activation/gating of calcium channels.


Patterns of expression of feline cytokeratins in healthy epithelia and mammary carcinoma cells.

Expression of keratins (cytokeratins, CK) in healthy feline epithelia and 2 established feline mammary carcinoma cell lines was examined immunohistochemically and by use of immunoblotting analysis. A panel of specific anti-CK monoclonal antibodies (MAb) identifying epitopes unique to individual keratins or shared by 2 (or 3) CK polypeptides was used. Besides already available anti-human CK MAb, this panel of MAb consisted of 9 newly generated anti-human CK MAb and 1 newly generated anti-feline CK MAb. Immunohistochemical analysis on normal epithelia revealed that most of the anti-human CK MAb and the anti-feline CK MAb reacted with both feline and human epithelia, with a comparable tissue distribution pattern. However, slight differences in CK tissue distribution pattern between human beings and cats were detected by one MAb. Immunoblotting analysis revealed that all anti-human CK MAb that were immunohistochemically reactive with feline tissues detected analogous CK in cats, indicating the presence of a number of common epitopes on human and feline CK. Two continuous cell lines derived from 2 distinct feline mammary adenocarcinomas, K248C and K266, were analyzed with respect to their CK phenotype. Although no difference in CK expression between the 2 cell lines was detected in vitro, a difference in CK phenotype was detected on subcutaneous transplantation of the 2 cell lines into nude mice. Although the K248C-induced adenocarcinomas maintained the same CK phenotype as observed in vitro, the CK pattern of the K266 heterotransplants, growing as adenosquamous carcinomas, changed with squamous differentiation. Our findings confirm the high degree of homology between mammalian CK, and on the basis of those findings, we suggest that CK proteins provide a set of markers valuable for the characterization of normal and neoplastic feline tissues and for studies of squamous metaplasia.


Effect of topical corticosteroids on the recovery of histamine releasing factors in nasal washings of patients with allergic rhinitis. A double-blind, randomized, placebo-controlled study.

Nasal washings (NW) have been used by many investigators as a readily available biologic fluid for studying the mechanism of allergic reactions. These fluids have been analyzed for the presence of various mediators, including cytokines. Recently, histamine releasing factors (HRF) have been detected in the NW. The objective of this study was to investigate the effect of treatment with topical corticosteroids on the recovery of these cytokines from the NW obtained from patients with allergic rhinitis. A group of 30 patients with ragweed pollen allergy were given either beclomethasone dipropionate (BDP) or placebo for 1 wk in a double-blind randomized manner. NW were performed twice before the start of the treatment period and were repeated twice at the end of the study. HRF activity was measured in the NW. Patients maintained a daily symptom score. The activity of HRF decreased significantly (mean +/- SD, pre = 37.2 +/- 21.3% versus post = 23.8 +/- 20.1%; p less than 0.01) in the BDP group, as did the mean symptom score (5.1 +/- 1.4 versus 1.5 +/- 1.5, p less than 0.01) at the end of the treatment period. In contrast, there was no significant change in HRF recovery (32.8 +/- 25.6% versus 33.8 +/- 25.3%; p less than 0.05) or symptom score (4.8 +/- 1.8 versus 5.4 +/- 1.9; p greater than 0.05) in the placebo group. There was a significant correlation between the net changes in symptom scores and the net differences in HRF activity. We speculate that the reduction in HRF in the nasal mucosa may contribute to the clinical efficacy of topical corticosteroids.(ABSTRACT TRUNCATED AT 250 WORDS)


Endoscopic laser therapy for obstructing and/or bleeding colorectal carcinoma.

The primary role of the neodymium yttrium aluminum garnet (Nd:YAG) laser has been to relieve obstruction and to control hemorrhage associated with malignant neoplasms throughout the gastrointestinal tract. In an initial series (IS), the authors demonstrated the efficacy of the Nd:YAG laser as initial preresectional therapy (PR) in 11 patients with obstructing and resectable left colonic or rectal tumors obviating initial operative diversion and allowing for primary resection and anastomosis. In addition, the authors have illustrated the benefit of the Nd:YAG laser in relieving obstruction and arresting bleeding in those patients with either widely metastatic or nonresectable (NR) locoregional disease. Their cumulative experience from 1985 to the present includes 53 patients (PR: 29 and NR: 24). In the PR group, 25 lesions were above the peritoneal reflection and 4 below. Twenty-five patients underwent low anterior resection and four abdominoperineal resection. In the NR group, 17 patients were treated for imminent obstruction and 7 for bleeding. Ten lesions were above and 14 below the peritoneal reflection. There was one laser-related complication in the series (1.8%). There was no significant morbidity or mortality in the PR group. The reduction in length of stay (LOS) and total hospital costs (THC), when compared to the IS has continued to be significant. Laser therapy for those patients in the NR groups is a safe and acceptable alternative to permanent colostomy with its accompanying morbidity and mortality.


Cholinesterases in the amyloid angiopathy of Alzheimer's disease.

Vessels affected by amyloid angiopathy in patients with Alzheimer's disease also displayed intense acetylcholinesterase and butyrylcholinesterase activity when examined by light and electron microscopy. The enzymatic properties of the vessel-bound cholinesterases were identical to those of the cholinesterases associated with senile plaques and neurofibrillary tangles. This cholinesterase activity is of unknown origin but represents one of the very few features common to all the major pathological markers of Alzheimer's disease.


Push-through intubation: effective palliation in 409 patients with cancer of the esophagus and cardia.

Between 1980 and 1989, 355 patients with cancer of the esophagus and 54 with cancer of the cardia underwent push-through intubation because of advanced tumor stage or medical contraindications to tumor resection. In 36 other patients (8.1%), the attempt at transtumoral intubation failed. The hospital mortality rate after intubation was 3.4%. The following complications were observed: hemorrhage in 2.0% of the patients, esophageal perforation in 4.9%, tube dislodgment in 12.7%, and tube obstruction in 4.4%. Early resumption of semisolid oral feeding was possible in 80% of the discharged patients. The actuarial 1-year survival rate was 7.7% and the median survival, 3.9 months. In conclusion, push-through intubation represents a valid therapeutic choice, which is indicated mainly for patients with a long, infiltrating, and circumferential stricture of the thoracic esophagus or cardia that is inoperable and for patients with an esophagorespiratory or esophagomediastinal fistula.


Cryopreserved allograft repair of aortic hypoplasia and interrupted aortic arch.

Mortality for interruption of the aortic arch approaches 100% within the first year of life if untreated. Prostaglandin E1 can stabilize the patient's condition in anticipation of surgical palliation, but total repair is required for long-term survival. Successful complete repair of type B interrupted aortic arch, hypoplasia of the left ventricular outflow tract, and ventricular septal defect was possible using a cryopreserved allograft in a child who previously had undergone unusual palliation.


[New data on the leukocyte functional morphology in pyo-septic processes].

It is established by electron microscopical radioautography that sepsis results in the lowering of neutrophils bacterial capacity while their absorptive capacity is retained. Living bacteria may be released from neutrophils undergoing degradation into the environment. The same is observed when phagocytized but still living bacteria are located in phagosomes which are fused with one another and with neutrophil plasma membrane; after the destruction of the latter bacteria are released into the extracellular space. This is a negative issue for the host of the incomplete phagocytosis. Neutrophils participate more actively in the lysis of the necrotic tissues and wound cleaning than in the bacteria phagocytosis.


Clinicopathologic correlation of occult choroidal neovascularization in age-related macular degeneration.

We report the clinicopathologic features of an eye with occult choroidal neovascularization associated with age-related macular degeneration. Ophthalmoscopic findings at presentation included subretinal fluid and lipid. We noted angiographic staining of irregularly elevated areas of retinal pigment epithelium. In the late phase of the angiogram, fluorescein leakage at the level of the outer retina was observed that did not correspond to well-demarcated areas of hyperfluorescence in earlier phases. The patient was randomized to treatment in a pilot trial comparing the effects of grid laser treatment with the effects of no treatment for occult choroidal neovascularization. Three weeks after treatment, some of the subretinal fluid had cleared and vision improved. The patient died 6 weeks after laser treatment. Histopathologic study disclosed a subretinal pigment epithelial fibrovascular membrane. Neovascularization originated from the choroid.


Transmission electron microscopic study of a subretinal choroidal neovascular membrane due to age-related macular degeneration.

From a patient with age-related macular degeneration we studied ultrastructurally a disciform scar that was removed from an eye with a vitreous hemorrhage. In cross section, the scar was divided by a retinal pigment epithelial (RPE) cell layer. The choroidal side consisted of fibrovascular tissue with active neovascular buds and inflammatory cells, including macrophages attached to the RPE basement membrane. Apart from the RPE, no components of Bruch's membrane could be identified. The retinal side contained organizing hemorrhage and a collagenous matrix with fibroblastlike cells probably of RPE and choroidal origin. The anatomy and the clinical findings at surgery suggest that such scars lie on (rather than within) the inner collagenous layer of Bruch's membrane and contain two components divided by the original RPE layer. The choroidal side is fibrovascular, including active neovascularization, and the retinal side is fibrous and formed by metaplastic RPE cells and choroidal fibrovascular ingrowth.


Observing development through evolutionary eyes: a practical approach.

An argument is made that only through a detailed comparison of mutational mechanisms underlying the evolution of the genetic systems governing development, can the 'logic' of individual development be fully comprehended. To do this, it is essential to choose two or more genes (or their products) that interact in the establishment of a given function, and to compare the molecular basis of that interaction in closely related species. The rationale to this approach arises from observations of molecular co-evolution between interacting partners involved with given functions which have led to species specificity in the manner in which such functions are effected. Molecular coevolution reveals that divergence in sequence can be tolerated whilst biological functions are maintained, not because it is neutral and dispensable but because successful, compensatory changes can evolve in eukaryotic genomes that are in continuous states of flux.


Binding of [3H]SCH23390 to a non-dopaminergic site in bovine kidney.

Binding of the D1 dopamine receptor antagonist [3H]SCH23390 to bovine renal cortical membranes has been studied. Specific binding of [3H]SCH23390 was saturable and reversible and stereoisomers of SCH23390 competed stereoselectively. In contrast, competition with the isomers of butaclamol was not stereoselective and dopamine failed to compete for the [3H]SCH23390 binding site. The site is therefore not a D1 dopamine receptor. Competition studies with a very wide range of compounds failed to define the nature of the [3H]SCH23390 binding sites in renal cortex whereas in parallel studies the characteristics of [3H]SCH23390 binding in caudate nucleus were entirely consistent with those of D1 dopamine receptors. The nature of [3H]SCH23390 binding in preparations of tubular and glomerular membranes was found to be virtually identical to those of crude renal cortical membranes indicating lack of compartmentation of these sites. Autoradiographic studies of [3H]SCH23390 binding in bovine kidney showed significantly higher levels of binding sites in renal cortex compared with renal medulla and this was confirmed by direct ligand binding studies.


Cell-cell contact mechanisms.

Exciting new findings link characteristic properties of the inflammatory process previously not linked functionally. For example, it is now clear that oxygen radicals and leukocyte adhesion are intimately related in a carefully transduced and orchestrated series of events that culminates in release of granule contents, but not before the leukocyte has safely transversed the vessel wall. In addition to defining separate heterocellular contacts and homocellular aggregation we must now consider equilibrium events that allow associations among cell-cell partnerships involving different cell types.


Coping with infertility: a new nursing perspective.

Infertility is a major life stressor, and reproductive failure is difficult to accept. Many couples become trapped in a cycle of repeated medical treatment, repeating painful patterns without developing insight or growth, feeling powerless to achieve their goal or move on. The causes of inappropriate coping behaviors are not well understood, and there is little direction for the nurse clinician's efforts to assist the couple with decision making. The use of superstition and ritual (magical thinking or magical ideation) by couples with infertility is discussed, and its role as a potential barrier to resolution is evaluated. A clinical tool for assessing the extent of magical ideation is described and its potential clinical utility explored. Women who have experienced a previous pregnancy may be more likely than nulliparas to substitute magical thinking for coping behaviors that might bring them closer to resolution. Problems with current research in this area are discussed, and directions for developing clinical nursing interventions are suggested.


[Nursing care of the patient with an artificial pacemaker].

The construction of high technology electronic artificial cardiac pacemaker and its application by the medical science has changed the way and the prognosis of the diseases of the cardiac conducting system. Nursing presence is essential in care of the patient with a cardiac pacemaker. In the operating theatre the nurse creates the most ideal environment and assists at the application of the artificial pacemaker; in the ward she observes with watchful glance for prevention of complications providing individual and holistic nursing care, and helps the patient and his family with rehabilitation and adaptation to his new way of life. In addition, the nurse teaches the patient how to observe the function of his pacemaker, and emphasizes the importance of keeping the doctor's orders and regularly visiting the outpatients cardiac pacemakers control department to check his pacemaker.


Intraocular gas effects on corneal endothelial permeability.

We examined the effects of intraocular gases on the permeability of the rabbit corneal endothelium to inulin and dextran. Volumes of air (0.16 ml), sulfur hexafluoride (SF6) (0.08 ml), and octafluoropentane (C3F8) (0.04 ml) were infused into the anterior chamber at constant intraocular pressure so that all volumes were equal after expansion. The inulin/dextran permeability was statistically decreased by infusion with Ringer, while air caused an 8.4% increase in dextran permeability but no effect on inulin flux. These small effects were of no biological significance. SF6 caused a 16% and 13% increase in inulin and dextran permeability, respectively, while C3F8 caused an 18% increase in both inulin and dextran permeability. Longevity of gas in the anterior chamber appears important in delineating the deleterious effects. The gases per se do not appear toxic but rather disrupt normal physiologic function through physical process.


Alterations in lens permeability during galactose cataract development in rat.

Lens permeability has been shown to be compromised during galactose-induced cataract development in rats. Recent studies have demonstrated permeability and diffusion pathways as well as endocytotic activity in normal lenses of different species using tracers of different molecular weights. We investigated the permeability and diffusion of tracers in normal rat lenses and in lenses during cataract development using three different molecular weight tracers, lanthanum nitrate (LN, MW 430), ruthenium red (RR, MW 858.5) and horseradish peroxidase (HRP, MW 40.000) for this study. Sprague Dawley rats weighing 50gms were fed Purina Rat Chow with or without galactose. Our results, based on transmission electron microscopy, show that all tracers penetrated through the capsule and the basal portion of the intercellular spaces. While the diffusion of RR was restricted to the epithelial cell layer, LN and HRP were observed in intercellular spaces in cortical fiber cells. In the HRP "washout" studies, HRP could be readily removed from the intercellular spaces in the basal region in the epithelial cell layer. This and other observations suggest the presence of a barrier (tight-junction) in the apical region. Our studies also suggest an influx of tracers in the lens, specifically LN and HRP, through the equatorial region. The permeability of the tracers increased and endocytotic vesicles with tracers were often found associated with the lateral and basal membranes of epithelial cells in the galactose-fed rats at the precataractous and mature cataractous stages. This study provides further support for the presence of a tight-junction between epithelial cells and indicates the movement of tracers through the equatorial region. Moreover, it confirms previous observations that indicated alterations in lens permeability during galactose cataractogenesis.


Carcinosarcoma of the breast. Immunohistochemical and ultrastructural studies.

A case of a carcinosarcoma in the breast of a 51-year-old woman is described. The tumor consisted of two diversely differentiated components with cells possessing epithelial and mesenchymal characteristics. Plenty of cytokeratin and CEA were immunohistochemically recordable from the carcinomatous component. Some neoplastic cells also stained positively for actin, S-100 protein and smooth muscle antigen. The sarcomatous component was positive only for vimentin. Electron microscopic analysis revealed presence of the following cell types: epithelial cells, myoepithelial cells and polymorphous mesenchymal cells.


Effects of diets containing casein and rapeseed on enzyme secretion from the exocrine pancreas in the pig.

The effect of dietary protein on enzyme activity of pancreatic juice was studied in ten growing, castrated, Large White male pigs. Animals, fitted with permanent cannulas in the pancreatic duct and in the duodenum, were divided into two groups receiving either casein or rapeseed concentrate as a protein source. After a 15 d adaptation period to the experimental diet, the volume of pancreatic secretion was significantly higher, whereas the protein concentration was lower in the casein group compared with the rapeseed group. No statistical difference was observed in the daily protein output between groups. Total secreted activities of carboxypeptidase A (EC 3.4.17.1), and elastase (EC 3.4.21.36) were higher in the casein group during the nocturnal period, whereas total activities of trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), carboxypeptidase B (EC 3.4.17.2) and amylase (EC 3.2.1.1) in pancreatic secretions during the post-prandial periods were increased by the ingestion of the rapeseed diet. It is concluded that the pancreatic enzyme secretion is sensitive to the nature of the protein ingested.


A PML/retinoic acid receptor alpha fusion transcript is constantly detected by RNA-based polymerase chain reaction in acute promyelocytic leukemia.

The t(15;17) translocation is specifically observed in patients with promyelocytic leukemia (AML3). The chromosomal rearrangement juxtaposes the retinoic acid receptor alpha (RAR alpha) and PML genes, resulting in PML/RAR alpha fusion transcripts. Our previous studies have shown that a polymerase chain reaction (PCR) amplification product could be obtained from the cDNA of the NB4 promyelocytic cell line from which the chimaeric PML/RAR alpha was cloned. We report here that in all 14 AML3 patients tested, reverse transcriptase-PCR (RT-PCR) allows the detection of three specific fusion products. In eight patients, one amplification product was detected corresponding to the previously described abnormal fusion. Five patients displayed a different amplified fragment corresponding to a different fusion point. One other patient always showed a third different-sized product. The different types of fusion transcripts amplified were correlated to the size of the abnormal RAR alpha transcripts detected in these patients by Northern analysis, but did not prove determinant for either the phenotypic features or the retinoic acid responsiveness in AML3 cells in this group of patients. The consistent identification by RT-PCR of the fusion of the PML and RAR alpha genes in AML3 patients suggest that this method will provide a useful tool for the diagnosis and detection of minimal residual disease in these patients.


Effective immunochemotherapy of CALLA+C mu+ human pre-B acute lymphoblastic leukemia in mice with severe combined immunodeficiency using B43 (anti-CD19) pokeweed antiviral protein immunotoxin plus cyclophosphamide.

A highly aggressive human CALLA+C mu+ pre-B acute lymphoblastic leukemia (ALL) cell line (NALM-6-UM1) causes disseminated and invariably fatal leukemia in CB.17 mice with severe combined immunodeficiency (SCID). We used this SCID mouse model of human pre-B ALL to evaluate and compare, in a total of 434 SCID mice, the antileukemic efficacy of B43 (anti-CD19)-pokeweed antiviral protein (PAP) immunotoxin and cyclophosphamide (CPA) as individual reagents and as combined immunochemotherapeutic regimens. B43-PAP plus CPA was superior to either the immunotoxin or drug alone, and combined immunochemotherapy markedly improved the event-free survival (EFS) of SCID mice challenged with NALM-6-UM1 pre-B ALL cells. Notably, 90% to 100% of SCID mice challenged with 1 x 10(6) leukemia cells and then treated with B43-PAP plus CPA combined immunochemotherapy regimens became long-term survivors, a result not achieved with B43-PAP alone or CPA alone. The advantage was particularly evident in mice inoculated with 5 x 10(6) leukemia cells. While neither 15 micrograms B43-PAP (median survival, 58 days) nor 1 mg CPA (median survival, 49 days) resulted in long-term EFS of SCID mice challenged with 5 x 10(6) NALM-6-UM1 pre-B ALL cells, the probability of EFS at 6 months was 50% +/- 16% for SCID mice treated with 15 micrograms B43-PAP plus 1 mg CPA (median survival, greater than 180 days) (P less than .0001). The probability of long-term EFS was only 14% +/- 7% for mice treated with 30 micrograms B43-PAP and 0% +/- 0% for mice treated with 1 mg CPA, but 40% +/- 16% for mice treated with 30 micrograms B43-PAP plus 1 mg CPA (P less than .0001). Similarly, the probability of EFS at 6 months was 40% +/- 16% for mice treated with 2 mg CPA alone, 70% +/- 15% for mice treated with 2 mg CPA plus 15 micrograms B43-PAP, and 70% +/- 15% for mice treated with 2 mg CPA plus 30 micrograms B43-PAP. Ten SCID mice in the B43-PAP plus CPA combined immunochemotherapy arms surviving long term after the inoculation of 5 x 10(6) NALM-6-UM1 pre-B ALL cells were electively killed at 174 to 181 days to assess their leukemia burden. We found no evidence of leukemia in any of the bone marrow specimens by two-color immunofluorescence and multiparameter flow cytometry.(ABSTRACT TRUNCATED AT 400 WORDS)


Further phenotypic characterization and isolation of human hematopoietic progenitor cells using a monoclonal antibody to the c-kit receptor.

A mouse antihuman monoclonal IgG2a antibody, termed stem cell receptor-1 (SR-1), specific for a determinant of the c-kit ligand receptor (KR), was used as an immunologic probe to analyze KR expression by human bone marrow hematopoietic progenitor cells. Monoclonal antibodies to CD34 and HLA-DR were used in a multicolor staining protocol in conjunction with SR-1 to further define the phenotypes of various classes of hematopoietic progenitor cells. Expression of KR (SR-1+) on hematopoietic progenitor cells identified subpopulations of cells expressing CD34 (CD34+). While one-half of the CD34- and HLA-DR-expressing cells (CD34+ HLA-DR+) expressed the KR (SR-1+), one-third of the CD34+ cells that lacked HLA-DR expression (CD34+ HLA-DR-) were SR-1+. The CD34+ HLA-DR+ SR-1+ cell population contained the vast majority of the more differentiated progenitor cells, including the colony-forming unit (CFU) granulocyte-macrophage; burst-forming unit-erythrocyte; CFU-granulocyte, erythrocyte, macrophage, megakaryocyte; and the CFU-megakaryocyte. The overall progenitor cell cloning efficiency of this subpopulation was greater than 31%. By contrast, the CD34+ HLA-DR- SR-1+ cell population contained fewer of these more differentiated progenitor cells but exclusively contained the more primitive progenitor cells, the BFU-megakaryocyte, high proliferative potential-colony-forming cell, and long-term bone marrow culture-initiating cell. The overall progenitor cell cloning efficiency of this subpopulation was greater than 7%. Both the CD34+ HLA-DR- and CD34+ HLA-DR+ cell subpopulations lacking KR expression contained few assayable hematopoietic progenitor cells. Long-term bone marrow cultures initiated with CD34+ HLA-DR- SR-1+ but not CD34+ HLA-DR- SR-1- cells, which were repeatedly supplemented with c-kit ligand (KL) and interleukin-3, generated assayable progenitor cells of at least 2 lineages for 10 weeks. These experiments demonstrate the expression of the KR throughout the hierarchy of human hematopoietic progenitor cell development. We conclude from our data that the KL and KR play a pivotal role in cytokine regulation of both the primitive and more differentiated human hematopoietic progenitor cells.


Biologic significance of constitutive and subliminal growth factor production by bone marrow stroma.

The "stromal" or adherent cells of long-term murine Dexter explant bone marrow cultures provide the best in vitro model of the bone marrow microenvironment. Colony-stimulating factor-1 (CSF-1) is produced constitutively by these cells and is easily detected, but most investigators have not found constitutive production of the other hemolymphopoietic cytokines. We have previously reported the detection of granulocyte-macrophage-CSF (GM-CSF) in murine stromal cultures and its induction by the lectin Pokeweed mitogen. The present studies analyzing stromal cytokine messenger RNA (mRNA) production by standard Northern blot analysis show constitutive production of mRNAs for CSF-1, GM-CSF, granulocyte-CSF (G-CSF), c-kit ligand (KL), and interleukin-6 (IL-6), but not IL-3, IL-4, or IL-5 by 3-week irradiated or nonirradiated murine Dexter stromal cells. Exposure of stromal cells to Pokeweed mitogen or IL-1 16 hours before RNA harvest induces the messages for GM-CSF, G-CSF, KL, and IL-6, but not IL-3, IL-4, IL-5, or CSF-1. Polymerase chain reaction amplification of cDNA made with reverse transcriptase from stromal RNA using two separate sets of IL-3-specific primers shows the presence of IL-3 message in irradiated stromal cells, which is only detectable with this more sensitive technique. The factor-dependent cell lines FDC-P1 and 32D are supported by the stromal cells without the addition of exogenous growth factors, demonstrating a cytokine activity in these cultures that is inhibited by the addition of anti-IL-3 or anti-GM-CSF antibodies. These data indicate that murine Dexter stromal cells constitutively produce CSF-1, GM-CSF, G-CSF, IL-6, KL, and IL-3. This growth factor production could explain the support of granulocyte, macrophage, and megakaryocyte production and stem cell maintenance in Dexter-type long-term murine bone marrow cultures.


Enhanced hematopoietic growth factor production in an experimental myeloproliferative syndrome.

The murine myeloproliferative syndrome induced by the myeloproliferative sarcoma virus (MPSV) has numerous similarities to human primary myelofibrosis. We have shown that medium conditioned by spleen cells of MPSV-infected mice has the capacity to support the growth of primitive blast cell colonies. The detection of this activity associated with MPSV infection stimulated us to characterize the hematopoietins responsible for this activity. Northern blot analysis showed a large increase, or induction, of interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage-CSF (CSF-1), and granulocyte-CSF (G-CSF) transcripts in the hematopoietic organs of MPSV-infected mice; however, no IL-3 transcript could be detected in either MPSV-infected or normal mice. Significant levels of IL-1 alpha, IL-6, G-CSF, and CSF-1 bioactivities were found in the serum of MPSV-infected mice, but not in controls. Additionally, analysis of medium conditioned by spleen cells of MPSV-infected mice showed the presence of tumor necrosis factor alpha bioactivity. The increased production of cytokines that are able to stimulate pluripotent hematopoietic stem cells corroborates the hypothesis of a possible involvement of hematopoietic growth factors in the development of some myeloproliferative disorders.


Stem cell factor stimulates the in vitro growth of bone marrow cells from aplastic anemia patients.

Aplastic anemia (AA) is a rare human bone marrow disorder of unknown etiology manifested by a strongly impaired growth of hematopoietic precursors. In this study, we examined the ability of recombinant human stem cell factor (SCF) to stimulate proliferation in vitro of bone marrow cells from 15 AA patients. All patients had been previously treated with antilymphocyte globulin (ALG). SCF, in combination with erythropoietin (Epo), interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF), increased the number of hematopoietic colonies formed in a semisolid medium by AA marrows. Maximal colony numbers reached 30% of the numbers observed with normal bone marrow cells. Proliferation of AA cells cultured in a liquid medium containing SCF together with Epo, IL-3, GM-CSF, and G-CSF approached 70% of the control level, as measured by 3H-thymidine incorporation. The effect of the combination of SCF with the other growth factors was more than 10 times stronger than that of the growth factors alone. The most marked effect of SCF was on the generation of erythroid colonies by precursor cells. The results demonstrate synergism between CSF and other hematopoietic growth factors, resulting in the most efficient stimulation of the in vitro growth of AA bone marrow cells described to date. Use of SCF, either alone or in combination with other factors, may be of potential value in treatment of AA.


Suppressive effect of granulocyte-macrophage colony-stimulating factor on the generation of natural killer cells in vitro.

We investigated the effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and recombinant human granulocyte-CSF (rhG-CSF) on the generation of natural killer (NK) cells in vitro. NK cells were cultured from selected human bone marrow cells obtained after the elimination of mature T and NK cells. rhGM-CSF significantly suppressed the generation of CD56+ cells and NK activity (P less than .01) in a dose-dependent manner. The generation of large granular lymphocytes (LGL) was also suppressed in the presence of rhGM-CSF (P less than .01). In contrast, rhG-CSF had no effect on LGL (P greater than .05). Both rhGM-CSF and rhG-CSF had no influence on the CD56+ cell count in the peripheral blood. These results suggest that rhGM-CSF suppresses the in vitro generation of NK cells.


CD16- CD56+ natural killer cells after bone marrow transplantation.

Natural killer (NK) cells are phenotypically defined as lymphocytes expressing the antigens CD56 and mostly CD16 (Fc gamma RIII), but lacking CD3. A small CD3- CD16- CD56+ NK cell subset has been described in normal individuals representing less than 2% of peripheral blood lymphocytes. We analyzed here 70 patients for their reconstitution of the immune system during follow-up after autologous or allogeneic bone marrow transplantation. In 35% of these patients, two different NK cell subsets, namely CD56+dim and CD56+bright cells, were observed. The mean duration of these two subsets after transplant was 4 months. Sixty-five percent of the patients exhibited an increased number of NK cells, but only the typical CD16+ CD56+dim population. The CD56+bright subpopulation represented a particular CD3- CD16- NK subset, with posttransplant frequencies up to 70% of all NK cells and 40% of peripheral blood lymphocytes, respectively. In contrast to normal CD56+dim NK cells, CD56+bright cells coexpressed the activation antigens p75 beta-chain of interleukin-2 receptor (IL-2R), CD2R, and CD26, but were negative for CD16. NK and antibody-dependent cellular cytotoxicity activity of CD56+bright cells was low compared with CD56+dim NK cells. But using IL-2 and interferon gamma, their cytotoxicity could be enhanced even more than in CD56+dim lymphocytes. These different subsets may reflect distinct activation or differentiation steps of NK cells during reconstitution of the immune system. Their differential response to IL-2 may be of functional importance for posttransplant cytokine therapy.


Clinical and prognostic significance of monoclonal small cells in the peripheral blood and bone marrow of various B-cell lymphomas.

Discordant lymphomas, in particular nodal large-cell lymphomas with marrow small-cell lymphoma, were discovered recently, and the prognosis of patients with such disease has been discussed. The small cells were reported to be small lymphocytic or small cleaved lymphoma cells. We have detected, by kappa-lambda imaging (KLI) with delta-curves, using a flow cytometer, small lymphoma cells in the peripheral blood (PB) and bone marrow (BM) of 41 untreated patients with various B-cell lymphomas expressing surface Ig (sIg+BCL), and evaluated their clinical and prognostic significance. Small cells were found in approximately 90% (37 of 41) of sIg+BCL patients when either PB or BM was analyzed and, overall, the presence of small cells correlated well with the disease activity. However, in some patients, a few cells remained in the PB (16%) or BM (27%) even when they were in remission, whereas in others, the cells were presented in the PB or BM several months before relapse. These results suggest that the detection of small cells in PB or BM by KLI may be helpful for screening and monitoring patients with sIg+BCL. When the patients were subdivided into three groups (normal, low, and high amplitude), according to the abnormal grade criteria of the delta-curves, which were based on the results of both PB-KLI and BM-KLI, the survival of the high-amplitude group tended to be shorter than that of the normal group (P = .068), which was particularly marked when the follow-up period exceeded 2 years. Moreover, as the group grading worsened (normal less than low less than high), the complete response rates deteriorated (100%, 71%, and 60%, respectively) and the respective relapse rates after complete remission increased (17%, 40%, and 67%). Thus, the determination of the proportion of small lymphoma cells in PB and BM by KLI may be useful for predicting the prognoses of patients with sIg+BCL.


Expression of the APO-1 antigen in Burkitt lymphoma cell lines correlates with a shift towards a lymphoblastoid phenotype.

APO-1 is a cell surface molecule that induces apoptosis when ligated with the monoclonal antibody anti-APO-1. Expression of APO-1 and response to anti-APO-1 was investigated in a number of Epstein-Barr virus (EBV)-positive and -negative Burkitt lymphoma (BL) cell lines, in EBV-immortalized lymphoblastoid cell lines, and in cells from fresh BL biopsies. APO-1 was not expressed in EBV-negative cell lines and in EBV-positive BL cell lines with a phenotype corresponding to BL tumor biopsy cells (CD10+, CD21-, CD23-, CD30-, CD39-, CDw70-, CD77+). Accordingly, fresh BL cells obtained from three BL biopsies were APO-1 negative. EBV-positive BL cell lines that had acquired a lymphoblastoid phenotype (CD10-, CD21+, CD23+, CD30+, CD39+, CDw70+, CD77-) upon prolonged in vitro cultivation, as well as normal B-lymphoblastoid cell lines, expressed a high density of APO-1. APO-1 may, therefore, be regarded as a B-cell activation marker. APO-1 expression is not the only prerequisite for anti-APO-1-induced apoptosis because 6 of 7 APO-1-expressing EBV-positive BL cell lines were not sensitive to anti-APO-1, whereas all lymphoblastoid cell lines were killed by anti-APO-1. The sensitivity of lymphoblastoid cell lines to anti-APO-1-mediated apoptosis may open a new therapeutic approach for the treatment of EBV-induced lymphoproliferative lesions in immunocompromised individuals, because these are composed of cells with a lymphoblastoid phenotype.


Deletion of the zinc-binding motif of CD13/aminopeptidase N molecules results in loss of epitopes that mediate binding of inhibitory antibodies.

The myeloid cell-surface glycoprotein CD13/aminopeptidase N (APN; EC 3.4.11.2) contains a pentapeptide (HExxH) in its extracellular domain that is characteristic of many zinc-dependent metalloproteinases. This region contains residues important for zinc binding and constitutes part of the catalytic domain of several metalloproteases. We deleted an internal fragment of 117 base pairs (bp) from the human CD13/APN cDNA, resulting in an in-frame deletion that included the sequences coding for this pentapeptide motif. The mutant cDNA was subcloned into a retroviral expression vector, and polypeptides encoded by the altered cDNA were expressed in transfected murine NIH-3T3 fibroblasts. The mutant CD13/APN molecules lacked enzymatic activity, and their intracellular processing to the cell surface was retarded by comparison with normal CD13/APN polypeptides. The mutant molecules also lacked epitopes required for binding of four of 19 CD13-specific monoclonal antibodies (MoAbs) tested in flow cytometric assays. Each of the four MoAbs also inhibited the enzymatic activity of wild-type APN molecules, suggesting that these antibodies may inhibit aminopeptidase activity by interfering with the enzyme's zinc-coordinating properties. Cells engineered to express mutant CD13/APN polypeptides at the cell surface provide a tool for defining the physiologic role of this enzyme on normal and malignant myeloid cells and marrow stromal cells.


Developmental hierarchy during early human B-cell ontogeny after autologous bone marrow transplantation using autografts depleted of CD19+ B-cell precursors by an anti-CD19 pan-B-cell immunotoxin containing pokeweed antiviral protein.

Sequential immunophenotypes of bone marrow (BM) and peripheral blood (PBL) lymphoid cells from 15 B-lineage acute lymphoblastic leukemia (ALL) patients who underwent autologous bone marrow transplantation (BMT) during complete remission were determined by dual-color immunofluorescence and multiparameter flow cytometry. Autografts were depleted of CD19+ B-cell precursors by an immunochemopurging protocol that combines B43-PAP, a potent anti-CD19 immunotoxin, and the cyclophosphamide congener 4-hydroperoxycyclophosphamide (4-HC). A marked interpatient variation was observed in the appearance and expansion of B-cell precursors repopulating the posttransplant marrow. The expression of CD10 and CD19 antigens during early B-cell ontogeny post-BMT preceded the expression of CD20, CD21, CD22, CD40, and sIgM. The surface antigen profiles of the emerging B-cell precursors were similar to those of fetal liver or fetal bone marrow B-cell precursors. Our comparisons of BM and PBL samples from patients in the early post-BMT period demonstrated that (1) PBL initially contains fewer B-lineage cells than does BM, and (2) circulating B-lineage lymphoid cells have a more mature immunophenotype than do BM B-lineage lymphoid cells. Comparison of the surface antigen profiles of day 30 versus day 100 or year 1 BM or PBL lymphoid cells showed an increase in the percentages of CD10+CD22- undifferentiated lymphocyte precursors, as well as CD19+sIgM- B-cell precursors (pre-pre-B), consistent with a time-dependent expansion of these B-cell precursor populations post-BMT. Importantly, the percentages of CD10+CD22+ and CD19+sIgM+ B-cell precursor (pre-B) populations also increased between 30 days and 1 year post-BMT, confirming the ability of emerging immature B-cell precursors to differentiate along the B-precursor pathway. The acquisition and expression of B-lineage differentiation antigens at different stages of the post-BMT B-cell ontogeny support the notion that the expression of these antigens is developmentally programmed. Similar to patients in previous autologous BMT studies, recipients of B-cell precursor-depleted autografts had normal or nearly normal serum immunoglobulin levels, suggesting that the maturing B-cell/plasma cell populations can produce and secrete immunoglobulins. The development of a functional CD19+ B-lineage lymphoid compartment in recipients of autografts which were depleted of CD19+ B-cell precursors corroborates the previously postulated existence of CD19- B-lineage lymphoid progenitor cells.


In vivo sensitization of human lung carcinoma to bleomycin by the cysteine proteinase inhibitor E-64.

We recently demonstrated that the cysteine proteinase inhibitor, E-64, sensitizes human Burkitt's lymphoma (Daudi) to the antitumor action of bleomycin (BLM) by blocking its metabolism. We now report that E-64 sensitizes the BLM resistant human lung carcinoma A-549 by a mechanism unrelated to the inhibition of BLM metabolism. Treatment of A-549 tumor-bearing nude mice with either BLM (10 mg/kg) or E-64 (40 mg/kg) every other day for 10 days did not inhibit tumor growth. However, a 30 min pretreatment with E-64 prior to BLM caused complete and sustained inhibition of tumor growth. In contrast to our results with Burkitt's lymphoma, E-64 did not inhibit BLM metabolism but rather enhanced the tumor accumulation of BLM; within 10 min of BLM administration, tumors from E-64 pretreated mice showed a 6-fold higher accumulation of BLM A2 compared to non-pretreated xenografts. Furthermore, the level of tumor-associated BLM A2 remained 2-fold higher in E-64 pretreated mice 20 and 30 min after BLM administration. In E-64 pretreated mice, the plasma level of BLM was increased by 2-fold. These results demonstrate that the cysteine proteinase inhibitor, E-64, sensitized human lung carcinoma A-549 to BLM and, contrary to the expected mechanism, this effect of E-64 was not related to the inhibition of BLM metabolism.


The effects of vesamicol on trains of endplate currents and on focally recorded nerve terminal currents at mammalian neuromuscular junctions.

1. The effects of vesamicol, an inhibitor of vesicular acetylcholine (ACh) storage, were studied on trains of endplate currents (e.p.cs) in the cut rat hemidiaphragm nerve-muscle preparation and on trains of focally recorded nerve terminal current waveforms in the mouse triangularis sterni nerve-muscle preparation. 2. In the rat, 0.1 and 1 microM (-)-vesamicol produced an enhancement of the rundown of e.p.c. amplitudes during trains of high frequency (50 Hz) nerve stimulation. However, 1 microM (+)-vesamicol had no effect on the rundown of e.p.c. amplitudes. 3. In the mouse, high concentrations of (-)-vesamicol (10-100 microM) produced a concentration- and stimulation-dependent decrease in the amplitude of the second negative-going deflection of focally recorded nerve terminal current waveforms. 4. At 1 mM, (-)-vesamicol produced a stimulation-independent decrease in the amplitude of the first negative-going deflection of the nerve terminal current waveforms, an increase in signal delay and evidence of nerve conduction failure. These all indicate a local anaesthetic-like block of nodal Na(+)-channels. 5. In contrast to its effects on trains of e.p.cs, the effects of vesamicol on the nerve terminal current waveforms were not stereoselective, the (+)-isomer being equipotent with the (-)-isomer. 6. Low concentrations of the Na(+)-channel blocking toxin, tetrodotoxin (15-60 nM), produced similar changes in the focally recorded nerve terminal current waveforms to those seen with vesamicol. 7. It is concluded that the stereoselective rundown of e.p.c. amplitudes produced by (-)-vesamicol is due to an effect, either direct or indirect, on ACh mobilization within motor nerve terminals. Furthermore, in mammalian species, the inhibitory effects of vesamicol on nodal Na+-channels which are seen at high concentrations do not contribute to the principal neuromuscular effects of the compound.


X-irradiation attenuates relaxant responses in the rabbit ear artery.

1. The relaxant actions of acetylcholine, substance P and calcitonin gene-related peptide, and the levels of neuropeptide Y and calcitonin gene-related peptide were assessed in the rabbit central ear artery 1, 4 and 6 weeks after a single dose of 45 Gy X-irradiation, a dose similar to that used clinically in intraoperative radiotherapy. 2. Relaxant responses induced by acetylcholine and substance P (both endothelium-dependent) and calcitonin gene-related peptide (endothelium-independent) were reduced, and endogenous neuropeptide Y and calcitonin gene-related peptide levels were unaffected after X-irradiation. 3. The mechanism(s) by which a single dose of 45 Gy X-irradiation may selectively damage relaxant, but not direct, contractile responses of the smooth muscle (as we have shown previously) of the rabbit central ear artery are discussed.


Involvement of capsaicin-sensitive neurones in the haemodynamic effects of exogenous vasoactive peptides: studies in conscious, adult Long Evans rats treated neonatally with capsaicin.

1. The regional haemodynamic effects of i.v. bolus injections of bradykinin (0.05 or 0.5 nmol), cholecystokinin (0.175 or 1.75 nmol), substance P (0.01 or 0.1 nmol) and calcitonin gene-related peptide (0.05 or 0.5 nmol) were assessed in conscious, adult Long Evans rats that had been treated neonatally with either capsaicin (50 mg kg-1, s.c.) or vehicle. 2. In vehicle-treated rats, both doses of bradykinin were without effect on blood pressure, but caused tachycardia and hindquarters vasodilatation. Moreover, after the higher dose there were dilatations in the renal and superior mesenteric vascular beds. In capsaicin-treated rats the hindquarters vasodilator effects elicited by both doses of bradykinin were significantly reduced, while the tachycardia and responses in the renal and superior mesenteric vascular beds were unchanged. 3. In vehicle-treated rats, cholecystokinin caused dose-dependent increases in blood pressure accompanied by renal, superior mesenteric and hindquarters vasoconstriction followed, after the higher dose, by a hindquarters vasodilatation. The lower dose produced a tachycardia, while there was a bradycardia followed by a tachycardia after the higher dose. In capsaicin-treated rats, the pressor response, as well as the renal vasoconstrictor effects of cholecystokinin, were greater than in vehicle-treated rats, while the heart rate, superior mesenteric or hindquarters responses were not different. 4. In vehicle-treated rats, substance P produced a dose-dependent depressor response and tachycardia accompanied by dilatations in the renal and hindquarters vascular beds and constriction in the superior mesenteric vascular bed. In capsaicin-treated rats, the responses to the lower dose of substance P were not different from those in vehicle-treated rats, while the depressor response to the higher dose of substance P was slightly less than in vehicle-treated rats and the renal vasodilatation was absent.5. In vehicle-treated rats, calcitonin gene-related peptide caused dose-dependent hypotensive and tachycardic effects associated with dilatations in renal and hindquarters vascular beds and a constriction in the superior mesenteric vascular bed. After the higher dose, the renal vasodilatation was followed by a modest vasoconstriction. In capsaicin-treated rats, the depressor responses to both doses of calcitonin generelated peptide were slightly more prolonged than in vehicle-treated animals, whereas the heart rate and renal and mesenteric vascular conductance changes were not significantly different. However, there was a more sustained hindquarters vasodilator response to the higher dose of calcitonin gene-related peptide in the capsaicin-treated rats.6. The results suggest that peripheral, capsaicin-sensitive neurones are involved in the cardiovascular responses to exogenous bradykinin and cholecystokinin in conscious rats. It does not appear that the extent of involvement of these neurones is underestimated on account of development of marked supersensitivity to the peptides they normally release, since responses to such peptides (e.g. substance P and calcitonin gene-related peptide) are relatively normal in capsaicin-treated rats.


The selective NK3 receptor agonist senktide excites a subpopulation of dopamine-sensitive neurones in the rat substantia nigra pars compacta in vitro.

Intra- and extracellular recordings were made from substantia nigra zona compacta neurones from an in vitro rat brain slice preparation. A subpopulation of dopamine-sensitive neurones were encountered which were potently excited by bath application of the NK3 receptor agonist, senktide. On these senktide-sensitive neurones, NK1 and NK2 receptor agonists were inactive. The excitatory action of senktide supports a role for tachykinins as putative neurotransmitters in the basal ganglia.


Role of muscarinic receptor subtypes in central antinociception.

1. The ability to modify the pain threshold by the two M1-muscarinic agonists: McN-A-343 and AF-102B and by the specific M2-agonist arecaidine was examined in mice and rats by using three different noxious stimuli: chemical (writhing test), thermic (hot-plate test) and mechanical (paw pressure test). 2. In the mouse hot-plate test McN-A-343 (20-50 micrograms per mouse i.c.v.) and AF-102B (1-10 mg kg-1 i.p.) produced significant antinociception which was prevented by atropine (1 microgram per mouse i.c.v.) and by the two selective M1 antagonists: pirenzepine (0.01 micrograms per mouse i.c.v.) and dicyclomine (0.08 micrograms per mouse i.c.v. or 10 mg kg-1 i.p.) but not by the specific M2-antagonist AFDX-116 (0.1 micrograms per mouse i.c.v.), naloxone (1 mg kg-1 i.p.) or by the acetylcholine (ACh) depletor hemicholinium-3 (HC-3) (1 micrograms per mouse i.c.v.). McN-A-343 and AF-102B were able to increase the pain threshold also in the mouse acetic acid writhing test and in rat paw pressure test. These antinociceptive effects were completely prevented by dicyclomine (0.08 micrograms per mouse i.c.v. or 10 mg kg-1 i.p.) but not by AFDX-116 (0.1 microgram per mouse or rat i.c.v.). 3. In contrast with the M1-agonists, the M2-agonist arecaidine (0.1-2 micrograms per mouse or rat i.c.v.) did not induce antinociception in all three analgesic tests. However, arecaidine, at the same i.c.v. doses, was able to reduce the pain threshold in the hot-plate and paw pressure tests.4. The site of muscarinic control of the pain threshold is localized in the CNS since drugs which do not cross the blood-brain barrier such as McN-A-343, pirenzepine and arecaidine exerted their effects only if injected i.c.v.5. On the basis of the above findings and existing literature we suggest that the postsynaptic muscarinic receptors involved in antinociception belong to the M1 subtype. Nevertheless, presynaptic autoreceptors (M2 subtype) may play a role in pain regulation since they are involved in modulation of endogenous ACh release.


Effects of acromelic acid A on the binding of [3H]-kainic acid and [3H]-AMPA to rat brain synaptic plasma membranes.

1. The ability of acromelic acid A to inhibit [3H]-kainic acid and [3H]-(RS)-alpha-amino-3-hydroxy-5-methyloxazole-4-propionic acid ([3H]-AMPA) binding to rat brain synaptic plasma membranes was investigated by equilibrium radioligand binding assay. 2. Kinetic analysis of [3H]-kainic acid binding demonstrated the existence of two kainate binding sites in this tissue preparation and yielded equilibrium dissociation constants for [3H]-kainic acid of KD = 0.4 nM and KD = 20.8 nM. 3. Kainic acid and domoic acid both appeared to displace [3H]-kainic acid from a single binding site with equilibrium binding constants of KD = 19.4 nM and Ki = 14.5 nM respectively. Acromelic acid A exhibited a biphasic inhibition of [3H]-kainic acid binding to synaptic membranes with binding affinities of Ki = 15.1 nM and Ki = 1.49 microM. 4. In the absence of chaotropic ions, the order of potency of inhibition of [3H]-AMPA binding was acromelic acid A (Ki = 26 nM) greater than AMPA (KD = 184 nM) greater than domoic acid (Ki = 499 nM). 5. The inclusion of 100 mM thiocynanate ion in the [3H]-AMPA binding assay resulted in a change in the order of potency to: AMPA (KD = 160 nM) greater than acromelic acid A (Ki = 289 nM) greater than domoic acid (Ki = 9.02 microM). 6. These results show that acromelic acid A distinguishes two kainate binding sites in rat brain synaptic plasma membranes and in addition, that in the absence of chaotropic ions, acromelic acid A is the most potent displacer of [3H]-AMPA binding yet described.


Projection neurons of the basolateral amygdala: a correlative Golgi and retrograde tract tracing study.

This study analyzed the projection neurons of the anterior subdivision of the rat basolateral amygdaloid nucleus (BLa) by correlating the morphology of Golgi-stained neurons with the morphology of neurons that were retrogradely labeled by injections into the main terminal fields of BLa. In each animal multiple injections of horseradish peroxidase (HRP) and wheat germ agglutinin-conjugated HRP were made into the prefrontal cortex and rostral striatum. These injections labeled approximately 85% of BLa neurons. The great majority of labeled neurons were the same shape and relative size as the pyramidal (class I) neurons described in previous Golgi studies. The unlabeled neurons appeared to correspond to the nonpyramidal (class II and class III) neurons described in Golgi studies. Thus this investigation provides experimental evidence that the pyramidal neurons are the main projection neurons of BL, whereas most of the nonpyramidal cells are local circuit neurons.


Effects of self- and cross-reinnervation on the numbers of motoneurons regenerating to forearm muscles in young and adult rats.

The present study was carried out to compare the ability of motoneurons to regenerate to functionally appropriate and inappropriate muscles, following axotomy at different stages of postnatal development. Five-, 10-, 21-day-old and adult rats of both sexes were used. In one group, the right median and radial nerves were cut and reunited. In a second group, the cut nerves were cross reunited and, in a third group the nerves were merely exposed. Following survival periods of up to one year, the extent of motoneuron regeneration through the repaired nerves was determined by injecting the retrogradely transported tracers horseradish peroxidase (HRP) and Fast Blue into the flexor and extensor muscles of the right forearm. The results were expressed in terms of the difference between the number of labelled motoneurons on the experimental side of the spinal cord and the number on the control side, the latter having been labelled by injection of HRP and Fast Blue into the muscles of the left forearm. Comparisons were then made between the groups with respect to the age at which axotomy occurred, and the target of regeneration. The results showed that when axotomy was performed in 5- and 10-day-old rats, significantly fewer motoneurons were labelled, irrespective of whether or not the target was functionally appropriate, than when axotomy was performed in adulthood. The difference was most likely due to a lower survival rate of motoneurons following axotomy in neonates. No difference was found, however, between the numbers of labelled median and radial nerve motoneurons following self- versus cross-reinnervation in any age group. This suggests that, in both adult and neonatal rats, motoneurons which survive axotomy are able to regenerate equally well to functionally appropriate or inappropriate muscles.


Brain dopamine D-2 receptor mechanisms in spontaneously hypertensive rats.

Brain dopaminergic function was studied in spontaneously hypertensive rats (SHR) with the selective dopamine D-2 antagonist sulpiride. Sulpiride dose-dependently inhibited locomotor activity of normotensive Wistar-Kyoto rats (WKY). SHR showed an increase in locomotor activity in response to low doses of sulpiride, whereas no effect was observed of higher doses. In a two-bottle salt-preference test, WKY showed increased preference for an 0.9% saline solution after treatment with sulpiride, whereas total fluid intake remained the same. In SHR, sulpiride influenced neither salt-preference nor total fluid intake. SHR and WKY with a unilateral lesion of the median forebrain bundle showed similar turning behaviour in response to treatment with amphetamine. Pretreatment with 100 mg/kg sulpiride virtually abolished amphetamine-induced turning in WKY, but had little effect in SHR. Sulpiride dose-dependently increased serum prolactin concentrations in WKY and SHR. However, the increase was significantly greater in SHR. Dopamine D-2 receptor binding was measured with in vitro autoradiography, using [125I]-sulpiride as the ligand. Binding density was similar in the caudate nucleus and substantia nigra of SHR and WKY brain. Concentrations of the dopamine metabolites DOPAC and HVA, but not of dopamine itself, were significantly increased in frontal cortex, striatum and hypothalamus after treatment with 100 mg/kg sulpiride. There were no significant differences between SHR and WKY in the increase in the DOPAC/DA and HVA/DA ratio. These data show that SHR show differential changes in their response to central dopamine D-2 blockade when compared to WKY. Thus, in some tests (locomotor activity after high doses, salt preference, turning behaviour), SHR respond less to sulpiride.(ABSTRACT TRUNCATED AT 250 WORDS)


Intensification of labelings of the immunogold silver staining method by gold toning.

We evaluated the applicability of the gold toning procedure to the immunogold silver staining method using monoclonal antibody against dopamine. Immunolabeling was examined in the rat substantia nigra at light and electron microscopic levels. Vibratome sections of fixed midbrains were incubated with anti-dopamine antiserum and then with 5 nm colloidal gold bound to goat anti-mouse immunoglobulin. Silver staining of these sections produced a light brown immunolabeling. After the sections were processed by gold toning, the labeling became intense black. At a light microscopic level, these high contrast signals were retained after the sections were osmicated and embedded in Epon. At an electron microscopic level, signal-to-noise ratio was high, and the positive staining could easily be verified at a low-power magnification. The technique described here, the gold-toned immunogold silver staining method, provides high contrast signals and is much more sensitive than immunogold silver staining alone. This method, therefore, has great potential for use in immunohistochemical analysis of the central nervous system.


Species differences in the immunophenotype of osteoclasts and mononuclear phagocytes.

Human osteoclasts, in contrast to mononuclear phagocytes, are known to express a well-defined restricted range of myeloid antigens. To determine whether these antigenic differences are present in other species, we examined the immunophenotype of chicken and rabbit osteoclasts, macrophages, macrophage polykaryons, and monocytes and compared them with similarly derived and cultured human cells. Human, rabbit, and avian osteoclasts reacted with monoclonal antibodies against human beta 1 integrins (CD29, CD49b, CD49d), beta 3 integrins (CD51, CD61), as well as human macrophage-associated antigen CD68. Avian osteoclasts also reacted for CD11a/18 and CD14 which are not present on human osteoclasts. Avian and mammalian monocytes, macrophages, and macrophage polykaryons expressed all the above antigens. Both avian and human macrophage polykaryons produced by culture of peritoneal macrophages reacted with anti-CD51 antibodies indicating that expression of the vitronectin receptor alone does not distinguish between these cells in vitro.


In vitro formation of mineralized nodules by periodontal ligament cells from the rat.

The purposes of this study were to determine whether periodontal ligament (PDL) cells are capable of producing mineralized nodules in vitro and to analyze ultrastructural features of the nodules. Rat PDL cells were obtained from coagulum in the socket at 2 days after tooth extraction and cultured at confluence in standard medium containing Dulbecco's Modified Eagle's Medium supplemented with 10% FBS and antibiotics. To test mineralized nodule formation, cells were further cultured for an additional 3 weeks in the standard medium containing (1) ascorbic acid (50 micrograms/ml) and sodium beta-glycerophosphate (10 mM), (2) ascorbic acid, sodium beta-glycerophosphate, and dexamethasone (5 microM), or (3) ascorbic acid alone. Cells were then fixed in 2.5% glutaraldehyde, postfixed in 1% OsO4, and prepared for light and electron microscopy. Three-dimensional nodules containing mineralized matrices were formed only when the cells were cultured in the presence of ascorbic acid and dexamethasone. They were composed of multilayered fibroblasts (up to 13 layers), and highly organized collagen fibrils with 64 nm cross-banding patterns between the cell layers. The fibroblasts in the nodules exhibited an elongated shape with a high degree of cytoplasmic polarity throughout the nodule, and have the morphological features of PDL fibroblasts as seen in vivo. Mineral deposition with needle-like crystals was initiated on collagen fibrils located in intercellular spaces of the upper cell layers and became increasingly heavier towards the bottom half of the nodules. X-ray microanalysis and electron diffraction analysis confirmed that mineral deposition contained calcium and phosphate in the form of immature hydroxyapatite.(ABSTRACT TRUNCATED AT 250 WORDS)


Metabolism and nucleic acid binding of N-hydroxy-4-acetylaminobiphenyl and N-acetoxy-4-acetylaminobiphenyl by cultured human uroepithelial cells.

Metabolic activation of N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) and N-acetoxy-4-acetylaminobiphenyl (N-OAc-AABP), the proximate carcinogenic metabolites of the human bladder carcinogen 4-aminobiphenyl (ABP), was examined in human uroepithelial cells (HUC). Bioconversion was studied by incubating HUC cultures with [3H]N-OAc-AABP or [3H]N-OH-AABP. Three organo-soluble metabolites, N-OH-AABP, 4-acetylaminobiphenyl (AABP), and ABP were identified in ethyl acetate extracts from cultures exposed to N-OAc-AABP. Similarly, AABP and ABP were characterized as the major metabolites from cultures treated with N-OH-AABP. Incubation of N-OAc-AABP with HUC microsomes in vitro yielded primarily the O-deacetylation product N-OH-AABP. The HUC microsomes also catalyzed the N-deacetylation of N-OAc-[14C]AABP, N-OH-[14C]AABP, and [3H]AABP. The O- and N-deacetylase activities for N-OAc-AABP were 55.9 and 38.2 nmol/mg/min, respectively. These O- and N-deacetylase activities were both blocked by paraoxon. Incubation of [3H]N-OAc-AABP or [3H]N-OH-AABP with HUC microsomes and tRNA or DNA showed that 23.0 and 8.0 nmol of N-OAc-AABP and 74.5 and 25.2 pmol of N-OH-AABP were bound per mg protein/mg RNA or DNA, respectively. In comparison, the acetyl CoA-dependent HUC cytosol-mediated bindings of [3H]N-OH-ABP to RNA and DNA were 801 and 447 pmol/mg nucleic acid/mg protein. The HUC microsome-mediated bindings of N-OAc-AABP and N-OH-AABP to nucleic acids were inhibited by paraoxon, whereas the cytosol-mediated binding of N-OH-ABP was insensitive to paraoxon inhibition. Chromatography of the DNA hydrolysate obtained from the in vitro incubation of [3H]N-OAc-AABP or [3H]N-OH-AABP with HUC microsomes showed N-(deoxyguanosine-8-yl)-4-aminobiphenyl as the major adduct, based on comparison with authentic synthetic standard. These results show that human uroepithelia contain microsomal acetyl transferases that are capable of converting the proximate metabolites N-OAc-AABP and N-OH-AABP of the human bladder carcinogen ABP, to reactive electrophiles that bind to DNA. The occurrence of these acetyl transferases in the target organ of the human bladder carcinogen ABP suggests that metabolic activation of some proximate metabolites of ABP could occur directly in HUC and could play a pivotal role in susceptibility to aryl-amine/acetamide induced human bladder cancers.


Estimation of prostatic growth using serial prostate-specific antigen measurements in men with and without prostate disease.

Prostate growth curves were estimated from serial prostate-specific antigen (PSA) measurements on frozen sera in three groups of men: (a) 16 men with no prostatic disease by urological history and examination; (b) 20 men with a histological diagnosis of benign prostatic hyperplasia (BPH) who had undergone simple prostatectomy; and (c) 18 men with a histological diagnosis of prostate cancer. The median number of repeated PSA measurements over an 8- to 26-yr period prior to histological diagnosis or exclusion of prostate disease was eight and 11 for noncancer and cancer subjects, respectively. Predicted rates of change in PSA (PSA velocity) were linear and curvilinear for control and BPH subjects, respectively. Subjects with cancer demonstrated both a linear and an exponential phase of PSA velocity. Based on time to double PSA, we estimated the epithelial doubling time for men without prostate disease to range from 54 +/- 13 yr at age 40 to 84 +/- 13 yr at age 70. For men with BPH, doubling times ranged from 2 +/- 13 yr at age 40 to 17 +/- 5 yr at age 85. Subjects with local/regional and advanced/metastatic cancer had similar PSA doubling times of 2.4 +/- 0.6 yr and 1.8 +/- 0.2 yr, respectively. These data are consistent with what is known about prostatic growth with age in men without prostate disease and BPH, and the kinetics of prostate cancer growth. Estimates of prostatic growth rate from changes in PSA may be useful clinically in management of men with prostate disease.


Growth inhibition by cholera toxin of human lung carcinoma cell lines: correlation with GM1 ganglioside expression.

The effect of cholera toxin (CT) on the growth of 12 small cell lung carcinoma (SCLC) and 15 non-small cell lung carcinoma (NSCLC) cell lines is presented. CT inhibited the growth of nine SCLC cell lines (concentration for 50% inhibition of growth, 27-700 ng/ml), all of which had abundant expression of GM1 ganglioside, the surface receptor for CT. CT-resistant SCLC all had greatly decreased GM1 expression. In contrast, CT inhibited the growth of only four of 15 NSCLC cell lines. Seven of the 11 CT-resistant NSCLC had levels of GM1 comparable to CT-sensitive NSCLC or SCLC. In a limited panel of cell lines, cyclic AMP (cAMP) agonists including forskolin, 8Br[cAMP], and dibutyryl[cAMP] did not consistently reproduce CT-mediated inhibition of cell growth, nor did these compounds overcome resistance of cells to the growth inhibitory effects of CT. Expression of the RI and RII regulatory subunits of cAMP-dependent protein kinase was similar in CT-resistant and CT-sensitive SCLC or NSCLC cell lines. In the presence of isobutylmethylxanthine, intracellular cAMP levels induced by CT in a CT-resistant, GM1(+) NSCLC cell line were comparable to those achieved in a CT-sensitive NSCLC cell line. We conclude that inhibition of lung carcinoma cell growth by CT in all cases requires expression of GM1, and in the case of SCLC cell lines the presence of GM1 is sufficient. In NSCLC cell lines, expression of GM1 is not sufficient for growth inhibition by CT. These findings imply refractoriness to growth inhibition by cAMP in GM1(+), CT-resistant NSCLC cell lines and the possibility of non-cAMP-related mechanisms for growth inhibition in CT-sensitive cell lines.


Dissociation of sensitivities to tumor promotion and progression in outbred and inbred SENCAR mice.

The sensitivity of outbred SENCAR mice and inbred SENCAR (SSIN) mice to multistage carcinogenesis was studied. Tumors were induced using either 7,12-dimethylbenz[a]anthracene or N-methyl-N'-nitro-N-nitrosoguanidine as initiators and 12-O-tetradecanoylphorbol-13-acetate or benzoyl peroxide as promoting agents. Although the number of papillomas per mouse was higher in SSIN than in outbred SENCAR mice, the number of carcinomas observed in the SSIN strain was significantly lower regardless of the initiator or promoter used. It was also observed that the expression of markers of premalignant progression (i.e., dysplasia, expression of keratin K13, and loss of keratin K1 expression) was markedly suppressed in SSIN papillomas. After 50 wk of promotion with 12-O-tetradecanoylphorbol-13-acetate, the pattern of expression of K13 and K1 in SSIN mice was comparable to the pattern observed in outbred SENCAR mice after 10 to 20 wk of promotion with 12-O-tetradecanoylphorbol-13-acetate. It was also observed that 67% of the tumors induced in SSIN mice by initiation with 7,12-dimethylbenz[a]anthracene exhibited a mutation in codon 61 of the Ha-ras-1 gene. This latter finding suggests that the differences observed in tumor progression between the inbred strain and the outbred stock are not related to a genetic alteration in the Ha-ras-1 gene but rather to an independent event that we have postulated to involve a putative suppressor gene. The data reported here suggest that the putative gene(s) that confers susceptibility to tumor promotion was segregated from the gene(s) involved in tumor progression during selection and inbreeding of the SENCAR mouse stock.


Recombinant human stem cell factor stimulates growth of a human glioblastoma cell line expressing c-kit protooncogene.

The growth of a panel of eight different human glioblastoma cell lines was examined in a human tumor cloning assay in agar, a tritiated thymidine uptake assay, and by counting cell numbers, in cultures performed in the absence or presence of increasing concentrations (1 to 100 ng/ml) of recombinant human stem cell factor (SCF). Growth of 7 of 8 cell lines was not significantly and reproducibly affected by recombinant human SCF. However, growth of the CRL 1620 cell line could be stimulated up to 5-fold by the cytokine. In contrast to the other cell lines investigated, CRL 1620 expressed the c-kit protooncogene assessed on the mRNA and protein level. Furthermore, SCF-induced proliferation of CRL 1620 cells was sensitive to the tyrosine kinase inhibitor erbstatin. Our data suggest that SCF can be operative in growth modulation of malignant cells outside the hematopoietic system, and this finding should be further studied for its possible clinical implications.


Rat macrophage activation after treatment with the bleomycin group of antitumour antibiotics in vivo.

We have previously reported that bleomycin and its derivative peplomycin enhance the release of cytokines by rat spleen cells during mitogen-stimulated cell culture in vitro, but liblomycin, another derivative of bleomycin, decreases cytokine release to below untreated control levels. Cytokine release correlated well with the inhibition of subcutaneous tumour growth after treatment with equivalent doses of the three analogues. In contrast, ascites tumour growth is completely inhibited by liblomycin and appears to be at least partly macrophage-mediated because the antitumour effect can be significantly inhibited by carageenan. This study shows that bleomycin and its analogues activate rat peritoneal macrophages and increase interleukin-6 release, O2- production, cell spreading, phagocytosis and random migration of macrophages, but only bleomycin enhances peritoneal macrophage invasion into a monolayer of rat lung endothelial cells in vitro. This study also shows that although liblomycin decreases spleen cell cytokine production and is less effective than bleomycin against subcutaneous tumour, as we have previously reported, the antitumour drug activates peritoneal macrophages and, compared to bleomycin, has a remarkable therapeutic effect on rat ascites tumour.


Influence of antibody protein dose on therapeutic efficacy of radioiodinated antibodies in nude mice bearing GW-39 human tumor.

The biodistributions of three 131I-labeled murine monoclonal antibodies, NP-4 and Immu-14 anti(carcinoembryonic antigen), and Mu-9 anti-(colon-specific antigen p), were determined at antibody protein doses varying from 1 microgram to 1000 micrograms in nude mice with small (0.1-0.4 g) GW-39 human colonic cancer xenografts. For each antibody, the percentage of the injected dose per gram of tumor and tumor/nontumor ratios were constant over a wide protein dose range. However, at high protein doses (above 100 micrograms for NP-4 and Immu-14) the percentage of the injected dose per gram of tumor and tumor/nontumor ratios decreased. Assuming that the uptake of a control anti-(alpha-fetoprotein) antibody represents the amount of antibody that accumulates in the tumor nonspecifically (i.e., antigen-independently), it could be shown that for each antibody the amount of antibody protein that accumulates in the tumor specifically, increases linearly with the protein dose, reaching a plateau level at the highest doses tested. The growth inhibition of GW-39 tumor transplants in nude mice treated with 131I-labeled antibody at either low or high antibody protein dose was compared. These experiments indicated that, in this experimental model, enhanced antibody protein dose may decrease the therapeutic efficacy of radioiodinated antibodies. It is suggested that heterogeneous distribution at low protein dose, with intense localization around the blood vessels, may enhance the tumoricidal effect of radioantibodies.


Recruitment of T lymphocytes and induction of tumor necrosis factor in thyroid cancer by a local immunotherapy.

To elucidate the mechanism of action for intratumoral injection of immunopotentiators, infiltrating mononuclear cells and tumor necrosis factor (TNF) were assayed by immunostaining tissue samples of differentiated thyroid cancer resected with or without presurgical local application of OK-432, a streptococcal preparation. Frozen sections of resected specimens were stained with monoclonal antibodies using either a conventional or a modified immunoperoxidase method. The tumors injected with OK-432 showed increased T lymphocyte infiltration and HLA-DR expression on cancer cells as compared to the non-injected controls. Among these T cells, the CD4+ subset was more numerous than the CD8+ population. In four out of the seven cases constituting the injected group, numerous TNF-positive cells were seen in clusters or lines as well as scattered, while none of the seven cases in the control group was associated with a considerable amount of these cells. In their morphology and distribution pattern, these TNF-positive cells appeared to be of macrophage lineage. Thus local injection of OK-432 in thyroid cancer was shown to recruit T lymphocytes of predominantly the CD4+ subset and to induce in situ production of TNF, a known potent tumoricidal cytokine. The present data warrant further studies in this direction besides wider clinical intratumoral application of the reagent.


Age-related changes of neuron-specific enolase, S-100 protein, and myelin basic protein concentrations in cerebrospinal fluid.

Studies on cerebrospinal fluid (CSF) concentrations of neuron-specific enolase (NSE), S-100 protein, and myelin basic protein (MBP) in patients with neurological lesions indicate a quantitative relation between the degree of cell damage in the central nervous system (CNS) and the concentration of these CNS-specific proteins in CSF. Thus NSE, S-100, and MBP could be of use as markers for destructive processes in the CNS. We collected 937 specimens of CSF from children and adults (from newborns to age 91 years) who were undergoing a diagnostic lumbar puncture for several clinical indications. Of these, 79 samples from subjects ranging in age from 0.7 to 66 years could be used retrospectively to construct a reference interval according to our criteria. In these 79 samples no sex dependency existed. The relative increase of NSE, S-100, and MBP with age was similar (1% per year), suggesting a common underlying mechanism. These results emphasize the necessity of using age-matched reference values when the CNS-specific proteins are to be evaluated in neurological diseases. We also present three case histories to discuss the possible clinical relevance of the measurement of NSE, S-100, and MBP in children and adults.


Two-site monoclonal antibody-based immunoradiometric assay for measuring prostate secretory protein in serum.

We developed a double-determinant immunoradiometric assay for measuring serum prostate secretory protein (PSP), using monoclonal antibodies (MAb) against two different epitopes: MAb PSP-19 was the capture antibody and MAb PSP-6 was the tracer antibody. Assay sensitivity was 0.1 microgram/L. Analytical recovery of PSP was 93.5-104.6%, whereas the intra- and interassay mean CVs were 4.2% and 6.9%, respectively. In 92 normal men, ages greater than 50 years, the mean PSP concentration was 5.7 micrograms/L, with 10 (10.9%) men having concentrations greater than 10 micrograms/L. In contrast, 20 of 49 (40.8%) patients with benign prostate hyperplasia (BPH; mean PSP concentration 9.4 micrograms/L) and 46 of 100 (46%) patients with prostate cancer (mean PSP concentration 22.2 micrograms/L) had PSP concentrations greater than 10 micrograms/L. Mean serum PSP concentrations of the BPH (P less than 0.05) and prostate cancer (P less than 0.01) groups were significantly different from those of age-matched normal men. In a small group of patients, serial PSP concentrations correlated with the clinical course during therapy. Thus, PSP may be a useful marker for evaluating patients with prostate cancer.


Immunoassays for quantifying choriogonadotropin compared for assay performance and clinical application.

We examined calibration and accuracy, precision, sensitivity, specificity, and "hook" effects for recently revised automated choriogonadotropin (hCG) immunoassay systems (Baxter-Dade Stratus II, Abbott IMx intact hCG and total beta hCG) and compared them with a widely used immunoradiometric assay (Hybritech). We estimated hCG in pregnant women, women with trophoblastic disease, nonpregnant young and menopausal women, normal men, and men with testicular tumors. We found clinically unimportant differences in calibration (all calibrated to the 3rd International Standard). Detection of hCG by all four assays was limited by their responses in serum from nonpregnant women and men. Precision within-run was best for the automated instruments, but all four assays had similar between-run precision. The Hybritech, Stratus, and IMx intact assays are specific for intact hCG. The IMx total beta assay quantifies both free beta subunit and beta subunit present in intact hCG. There is a clinically important hook effect in the Hybritech assay but not the Stratus or IMx assays (to 1.2 x 10(6) int. units/L). Results for pregnant women were similar by all four assays. We measured "hCG" to 8 int. units/L in menopausal women, which weakly correlated with concentrations of lutropin and follitropin and was, in part, explained by crossreactivity. There was no sample-probe carryover in either instrument. We found the IMx diluting module as well as results at the extremes of the IMx calibration curves (less than 10, 800-1200 int. units/L) unreliable but encountered no such problems with the Stratus system. Both automated systems involve batch analyzers with limited throughput but provide hCG concentration estimates much more quickly than the Hybritech assay can.