Sensitivity of B16 melanoma sublines to lymphokine-activated killer cells as determined by 51Cr-release and clonogenic assays

The aim of this study was to compare the differential sensitivities of B16 melanoma sublines to LAK cells by means of the standard 51Cr release assay and a clonogenic assay, which measures both cell survival and proliferation. LAK cells, generated after 4 days incubation with 150 international units (IU)/ml of interleukin-2 (IL-2), showed both cytolytic and anti-proliferative activities against B16 targets. Using an 18 h 51Cr release assay, murine LAK cells showed the highest cytolytic activity against B16 parental cells compared to B16-F1, B16-F10, B16-FLR and B16-BL6 sublines at effector/target (E/T) ratios ranging from 6/1 to 100/1. Purified adherent LAK (A-LAK) cells showed greater cytolytic activity against B16 parental cells and other B16 sublines compared to LAK cells, but otherwise the pattern of reactivity was similar. Using a clonogenic assay, the surviving fraction of B16 parental cells co-cultivated with LAK cells decreased to 0 at an E/T ratio of 50/1, while a 400/1 ratio was required to achieve a similar reduction of B16-F1, B16-F10, B16-FLR, and B16-BL6 sublines. No differences in subline sensitivity were seen with the 51Cr release assay, but these were observed using the clonogenic assay. An inverse linear relationship existed between % surviving fraction, as determined by the clonogenic assay, and cytolytic activity, as determined by the 51Cr release assay. Our data indicate that the clonogenic assay can detect differences in target cell sensitivity that otherwise are undetectable by the standard 51Cr release assay. The clonogenic assay may prove useful in delineating the long-term anti-adherent and anti-proliferative properties of effector cells from their cytolytic activity.     

Phase I clinical and pharmacokinetic trial of dextran conjugated doxorubicin (AD-70, DOX-OXD)

Coupling of anthracyclines to high-molecular-weight carriers may alter drug disposition and improve antitumor effects. We have performed a clinical phase I trial of doxorubicin coupled to dextran (70000 m.w.). The drug was administered as single dose i.v. every 21–28 days. Thirteen patients have received a total of 24 courses (median 2; range 1–3). At the starting dose of 40 mg/m² doxorubicin equivalent (DOXeq), WHO grade IV thrombocytopenia was noted in 2/2 patients. WHO grade IV hepatotoxicity and WHO grade III cardiotoxicity were noted in a patient with preexisting heart disease. Five patients were treated with 12.5 mg/m² DOXeq. Maximal toxicity at this dose level was WHO grade III thrombocytopenia and local phlebitis (WHO grade II) in 1/5 patients, elevation of alkaline phosphatase (WHO grade III) and WHO grade III vomiting in another patient. Subsequently, five patients received 20 mg/m² DOXeq. Hepatotoxicity was noted in 5/5 patients (1 × WHO grade IV, 1 × WHO grade III). Thrombocytopenia was noted in 3/5 patients (1 × WHO grade IV, 2 × WHO grade III). At 12.5 mg/m² DOXeq, a patient diagnosed with a malignant fibrous histiocytoma had stable disease for 4 months. Pharmacokinetic analyses of total and free doxorubicin were performed in plasma and urine. The maximum peak plasma concentration (ppc) for total DOX was 12.3 g/ml at 40 mg/m² DOXeq. The area under the plasma concentration time curve (AUC) ranged from 28.83–80.21 g/ml^*h with dose-dependent elimination half lives (t_1/2: 0.02–0.87 h;_1/2: 2.69–11.58 h;_1/2: 41.44–136.58 h). We conclude that the maximal tolerated dose (MTD) of AD-70 using this schedule is 40 mg/m² DOXeq. The recommended dose for clinical phase II studies is 12.5 mg/m² DOXeq.Abbreviations ALT Alanine Aminotransferase - AST Aspartate Aminotransferase - DOX Doxorubicin - DOXeq Doxorubicin Equivalent - ECG Electrocardiogram - HPLC High Pressure Liquid Chromatography - LD₁₀ Lethal Dose for 10% of individuals - MTD Maximal Tolerated Dose - ppc Peak Plasma Concentration - WHO World Health Organisation     

Characterization of four newly established human colorectal adenocarcinoma cell lines from Chinese patients.

Four colon adenocarcinoma cell lines, CC-M2, CC-M3, CC-M4, and CC-M2NM, have been established from surgical specimens of 18 unselected patients without the use of "feeder" cells and additional growth factors (e.g., insulin, hydrocortisone, etc.) in the culture medium. The methods of primary cultivation of tissue explants are described. Studies of determination of morphology, growth curve, plating efficiency, chromosomal analysis, CEA and beta-HCG synthesis, and tumorigenicity, were done to characterize the cell lines. Significant variations have been found in one of the four cell lines, both in vitro and in vivo studies. There are distinct phenotypes in the established cell lines which may be useful in studying the cell differentiation and progression of colorectal cancer.     

Differential effects of zidovudine and zidovudine triphosphate on mitochondrial permeability transition and oxidative phosphorylation

1. The effects of zidovudine (ZDV) and zidovudine triphosphate (ZDV-3P) on Ca²⁺-induced mitochondrial permeability transition (MPT), respiratory control ratio (RCR) and ATP synthesis have been investigated on isolated rat liver mitochondria.
2. ZDV slightly but significantly decreased RCR and ATP synthesis but was ineffective in inhibiting MPT. In contrast, ZDV-3P did not alter RCR and ATP synthesis but strongly inhibited MPT (IC₅₀=3.0±0.9 μM).
3. The effect of ZDV-3P on mitochondrial swelling required a preincubation time. When incubated 10 min with mitochondria, ZDV-3P (8 μM) totally inhibited the rate of swelling.
4. ADP, ATP and atractyloside, which are agents known to interact with the mitochondrial adenine nucleotide carrier (ANC), antagonized the effect of ZDV-3P on mitochondrial swelling. Indeed, the IC₅₀ value of ZDV-3P increased from 3.0 to 17.4, 93.6 and 66.5 μM, in the presence of 20 μM, ADP, ATP or atractyloside, respectively.
5. ZDV-3P did not displace [³H]-ATP from its mitochondrial binding site(s) whereas ADP and atractyloside did, suggesting that ZDV-3P and [³H]-ATP do not share the same binding sites.
6. ZDV-3P did not affect either mitochondrial respiration or ATP synthesis but inhibited Ca²⁺-dependent mitochondrial swelling. It was concluded that mitochondrial toxic effects observed during the chronic administration of ZDV cannot be related to its active metabolite (ZDV-3P).

     

Relationship of amyloid β/A4 protein to the neurofibrillary tangles in Guamanian parkinsonism-dementia

The Chamorro population of the island of Guam is highly susceptible to a disease called lytico-bodig (LB), wich clinically resembles a mixture of amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer disease (AD). The disease is characterized by the widespread development of neurofibrillary tangles in the central nervous system. These tangles have an immuno-histochemical profile indistinguishable from that seen in AD. We studied by immunohistochemistry the occurrence of intracellular and extracellular neurofibrillary tangles in LB in the entorhinal cortex, hippocampus and substantia nigra using antibodies to tau protein and ubiquitin. We also studied the relationship of these tangles to amyloid precursor protein (APP) and its -amyloid fragment (BAP), using multiple antibodies to BAP and other APP sequences. In advanced cases of LB, the development of neurofibrillary tangles was far more severe than in advanced cases of AD. Virtually all neurons of CA-1 and the subiculum were lost and only ghost tangles remained. In areas dominated by such extracellular tangles, BAP deposits were frequently observed developing around the fibers of ghost tangles. In some cases, the deposits covered only a few of the fibers, but in others, they seemed to envelope the complete tangle. The deposits were thiolavin S and Congo red positive, indicating that the BAP was in a consolidated form. We describe these entities as tangle-associated amyloid deposits. Such BAP deposits have previously eeen described in some cases of AD, dementia pugilistica and LB. However, we found them in all cases of LB with dementia in the hippocampal-entorhinal areas and in most cases in the substantia nigra. They do not evolve from diffuse BAP deposits since they are remote from them, and they do not trap dystrophic neurites. The fact that extracellular tangle material can act as a nidus for BAP build-up in LB suggests that further consideration needs to be given to the ways in which extracellular BAP deposits are formed.     

Early desensitization of somato-dendritic 5-HT1A autoreceptors in rats treated with fluoxetine or paroxetine

Electrophysiological and autoradiographic approaches were used to assess possible changes in 5-hydroxytryptamine (serotonin) 5-HT_1A receptors in the rat dorsal raphe nucleus after a subchronic treatment with fluoxetine or paroxetine, two specific serotonin reuptake inhibitors with antidepressant properties. Fluoxetine or paroxetine were injected daily (5 mg/kg, i.p.) for various time periods up to 21 days. Electrophysiological recordings performed 24 h after the last injection showed that the potency of the 5HT_1A receptor agonist, 8-OH-DPAT, to depress the firing of serotoninergic neurons in the dorsal raphe nucleus within brain stem slices was significantly reduced as early as after a 3-day treatment with either drug. The proportion of recorded neurons showing desensitization of somatodendritic 5-HT_1A autoreceptors increased along the treatment from 40% on the 3rd day to 60–80% on the 21st day. At no time during the treatment, was the specific binding of [³H]8-OHDPAT (agonist radioligand) or [³H] WAY-100 635 (antagonist radioligand) to 5-HT_1A receptors modified in the dorsal raphe nucleus or in other brain areas, suggesting that neither the density nor the coupling of these receptors to G-proteins were probably altered in rats injected with fluoxetine or paroxetine for up to 21 days.These results show that adaptive desensitization of somatodendritic 5-HT_1A autoreceptors within the dorsal raphe nucleus can already be detected after a 3-day treatment with selective serotonin reuptake inhibitors. Rather than the desensitization per se, it may be the progressive increase in the number of serotoninergic neurons with desensitized 5-HT_1A autoreceptors which plays a critical role in the (slowly developing) antidepressant action of these drugs.     

Native Hawaiian Birth Weight and Infant Mortality: Is Birth in Hawai'i Protective?

PURPOSE OF THE PAPER: This study provides baseline information on the characteristics of Native Hawaiian mothers and the health status of their infants, comparing residents of Hawaii with those of the continental U.S. The impact of Hawaii residence on low birth weight and infant mortality among Native Hawaiians is assessed. SUMMARY OF METHODS UTILIZED: Data from the National Center for Health Statistics 1983­1987 Linked U.S. Live Birth and Infant Death file were used to examine parental characteristics, prenatal care use and infant outcomes using chi­square and logistic regression procedures. PRINCIPAL FINDINGS: Despite a higher sociodemographic risk profile among Hawaii resident mothers, preterm birth, low and very low birth weight percentages were similar. Continental infants had significantly highter percentages of very pre­term birth and macrosomia. Mortality rates in both the neonatal and post­neonatal periods, and for SIDS and perinatal causes were elevated among continental infants. Hawaii residence had a borderline protective effect on infant mortality, wehn sociodemographic and prenatal care differences were controlled. CONCLUSIONS: This study suggests a possibly protective effect of Hawaii residence on the health of Native Hawaiian infants during the period of following employer­mandated health insurance coverage but before the initiation of "gap­group" coverage and the Native Hawaiian Health Care Systems in Hawaii. RELEVANCE TO ASIAN PACIFIC ISLANDER AMERICAN POPULATIONS. This is the first report documenting the sociodemographic and health status of the growing number of Native Hawaiian mothers and their infants residing outside of Hawaii. Expanded health insurance coverage and culturally appropriate and accessible health care may contribute to improved infant health status in Hawaii. Their absence, along with possible barriers of sociocultural isolation, may account for the poorer than expected outcomes of continental infants and predict a widening gap between them and their counterparts in Hawaii. A follow­up study of the health status of Native Hawaiian mothers and infants, and their access to appropriate care in Hawaii and thei continental U.S. is recommended.     

Hemispherectomy — 25 years later — findings and concepts

Summary In the first part the case of a then 27 year old female patient with right-sided infantile spastic hemiplegia after left-sided porencephaly is described, in whom hemispherectomy was performed 25 years ago. The postoperative state and the development are described as well as the social outcome and the present neurological status. A computer-tomogram shows the actual state of the brain.The second part is devoted to a scientific discussion of the supplementary motility after pyramidal lesions which is defined as the action of descending subcortico-spinal pathways starting probably in the mesencephalon, whereas an ipsilateral pathway is unlikely. Comparative neurological experiments serve to support such a concept as well as similar observations in traumatic cerebral lesions in man. In the light of the far more skilled motility in cerebral lesions of the young as well as the possibility of a shift of neuropsychological functions of the dominant to the contralateral hemisphere in children up to the age of 8–10 the possibility is discussed that plasticity — the concept of Albert Bethe — could form the mechanism of auxiliary function.Furthermore the surprising sensory performances in some of the patients with hemispherectomy are emphasized and the possibilities of anipsilateral substratum are discussed; however, this contrasts with the concepts formulated for the auxiliary motility after pyramidal lesions. Clarification of these problems will probably follow only after further experimental work.

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Zusammenfassung Im ersten Teil der Arbeit wird der Fall einer damals 27-jährigen Patientin beschrieben, die eine rechtsseitige infantile spastische Hemiplegie nach linksseitiger Porencephalie hatte. Bei ihr war 25 Jahre zuvor eine Hemisphärektomie durchgeführt worden. Der postoperative klinische Status und die Entwicklung der Syndrome wird beschrieben und über die soziale Situation berichtet. Schließlich wird der derzeitige neurologische Status genau wiedergegeben. Erstmalig zeigt ein Computer-Tomogramm den Zustand des Hirns nach diesem Zeitraum.Im zweiten Teil der Arbeit wird die Entstehung der Ersatz-Motilität diskutiert, die als eine Aktion deszendierender subkortiko-spinaler Bahnen definiert wird, die wahrscheinlich vom Mittelhirn ihren Ausgang nehmen, während die Tätigkeit ipsilateraler Systeme für unwahrscheinlich gehalten wird. Vergleichende Tierexperimente am Hirn unterstützen eine solche Auffassung, ebenso wie auch die Beobachtung bei umschriebenen Hirnverletzungen des Menschen.Dann wird die Möglichkeit von Ersatzfunktionen durch Plastizität diskutiert — ein Begriff von Albert Bethe. Dies erscheint im Lichte vieler Beobachtungen wichtig, da die Ersatz-Motilität bei Hirnläsionen junger Menschen sehr viel höhere Geschicklichkeit ermöglicht als bei Läsionen von Älteren; weiter ist zu beachten, daß neuropsychologische Funktionen bei einer Hirnläsion vor dem 8. bis 10. Lebensjahr von einer dominanten Hemisphäre noch auf die andere Seite verlegt und dort lokalisiert werden können. Schließlich wird bei diesen Patienten auf die erstaunlichen Fähigkeiten der sensiblen Systeme nach Hemisphärektomie hingewiesen und hier die Möglichkeit eines ipsilateralen anatomischen Apparates diskutiert. Jedoch können bei der Sensibilität so genaue funktionell-morphologische Vorstellungen noch nicht formuliert werden, wie sie sich z. Zt. schon für die Ersatz-Motilität nach Pyramidenbahnläsion ergeben. Erst genauere Experimente im Tierversuch können weitere Aufklärung bringen.

     

Septo-optic dysplasia and growth hormone deficiency: accelerated pubertal maturation during GH therapy

We report four patients (three male, one female) with septo-optic dysplasia and growth hormone deficiency. All had GH therapy for a period of four to eight years until reaching final height. In all four cases bone maturation during puberty was accelerated (1.4 to 1.9 "years"/year), resulting in a final height which was clearly below the predicted height. The progress of pubertal stages was very short in all patients. In three patients TSH and prolactin release after TRH stimulation were increased. These data support a hypothalamic original of the endocrine disorder. Insufficient GH release, even after repeated GHRH stimulation, is in contrast to this assumption. In one case there was a late manifestation of neurohormonal diabetes insipidus, which indicates the possibility of later disease progression. MR imaging of the brain demonstrated variable malformation of the septum pellucidum, chiasma and nervus opticus or the pituitary gland, respectively.