Specific eradication of micrometastases by transfer of tumour-immune T cells from major-histocompatibility-complex congenic mice

Summary DBA/2 (H-2^d) mice bearing a transplanted highly metastatic lymphoma (ESb) in a state of widely disseminated disease could be successfully treated by a combination of surgery (removal of the local tumour), irradiation (5 Gy) and adoptive immunotherapy. The immunotherapy was achieved by transfer of anti-ESb-immune spleen cells from B10.D2 mice, which express the same major histocompatibility complex (MHC) molecules as DBA/2. In contrast, anti-ESb-immune cells from MHC-disparate C57BL/6 mice did not confer protective immunity. The B10.D2 anti-ESb-immune T cells contain two types of cytolytic specificity as detected by limiting-dilution analysis: (1) clones with specificity for the ESb-tumour-associated transplantation antigen (TATA) (at low frequency), and (b) clones with specificity for minor DBA/2 histocompatibility (H) antigens (at high frequency). Immune B10.D2 cells raised against different tumour lines or against TATA^– ESb tumour variants did not confer the 100% protection seen with immune cells against ESb TATA⁺ cells. Finally we demonstrate that the allogeneic immune cells are more potent in terms of protective immunity than corresponding syngeneic immune cells. The data suggest that the strong graft-versus-leukemia effect with immune T cells from allogeneic MHC-identical but not from MHC-disparate mice was due to T cells with MHC-restricted specificity for an ESb-associated TATA. A graft-versus-host reactivity that developed much later and could not be prevented was most likely due to T cells sensitized against normal minor H antigens of the host. Our results are of potential relevance for allogeneic bone marrow transplantation and adoptive immunotherapy protocols.     

Induction of lymphokine-activated killer activity in rat splenocyte cultures: The importance of 2-mercaptoethanol and indomethacin

Summary The role of 2-mercaptoethanol and indomethacin in the induction of lymphokine-activated killer (LAK) activity by interleukin-2 (IL-2) in rat splenocyte cultures was investigated. Spleens from 4-month-old male rats of five different strains were tested. Splenocytes were cultured for 3–5 days in the presence of IL-2 (1000 U/ml) and LAK activity was assessed by 4-h⁵¹Cr release assays with P815 and YAC-1 cells as targets. LAK activity could be induced by IL-2 in splenocytes from all rat strains, but only when 2-mercaptoethanol was present in the culture medium. Optimal LAK activity was induced when the 2-mercaptoethanol concentration in splenocyte cultures was at least 5 µM. Different rat strains showed differences in levels of in vitro induction of LAK activity. In the presence of 2-mercaptoethanol the level of LAK activity induced by IL-2 was high in BN and Lewis rats, intermediate in Wistar and Wag rats, and low in DZB rats. In the absence of 2-mercaptoethanol no or minimal LAK activity was induced. Furthermore we observed that addition of 50 µm indomethacin to the culture medium in the presence of 2-mercaptoethanol augmented the induction of LAK activity to some extent. In the absence of 2-mercaptoethanol, addition of indomethacin resulted only in low levels or no induction of LAK activity. We conclude that for optimal induction of LAK activity by IL-2 in rat splenocyte cultures 2-mercaptoethanol is essential, while indomethacin can only marginally further improve this induction.     

Bacteriophage receptors of Lactococcus lactis subsp. 'diacetylactis' F7/2 and Lactococcus lactis subsp. cremoris Wg2-1

Bacteriophage P008 revealed irreversible and uniform adsorption to cell walls of L. lactis subsp. 'diacetylactis' F7/2, whereas phage P127 adsorbed reversibly to a limited number of receptor sites on cell walls of L. lactis subsp. cremoris Wg2-1. Neither extraction of lipids, cell wall- and membrane-teichoic acids nor enzymatic degradation of proteins altered the binding efficiencies of both cell wall fractions. However, phage binding was inhibited, when cell walls were subjected to lysozyme, metaperiodate, or acid treatments. This reflects that a carbohydrate component embedded in the peptidoglycan matrix is part of the phage receptors of strains F7/2 and Wg2-1.     

Isolation and characterization of two proteoheparan sulfate species of calf arterial tissue

When calf aortic tissue, preincubated under organ culture conditions in the presence of [35S]sulfate, was submitted to a sequential collagenase and elastase digestion and guanidinium chloride extraction, the bulk of proteoheparan sulfate was obtained in the elastase fraction. Ion-exchange chromatography on DEAE-cellulose of the elastase digest under dissociative conditions yielded a proteoglycan fraction that contained heparan sulfate as the sole glycosaminoglycan. The proteoheparan sulfate fraction was resolved into a high-molecular-mass (P-HS 1) and a low-molecular-mass (P-HS 2) fraction by gel filtration on Sephacryl S-400. P-HS 1 has a Mr of 175,000 and possesses four heparan sulfate side-chains (Mr 32,000) covalently bound to the protein core via a galactose- and xylose-containing polysaccharide-protein binding region. The protein core (Mr 38,000), which was obtained after deglycosylation of PG-HS 1 with trifluormethane sulfonic acid, contained in addition a few N-glycosidically linked oligosaccharide units representing a complex type with terminal neuraminic acid residues. P-HS 2 is a single-chain peptidoheparan sulfate of Mr of 38,000 containing one heparan sulfate chain (Mr 32,000) linked to a polypeptide (Mr 6000). The ratio of specific radioactivities of P-HS 1 and P-HS 2 was 1:0.66.     

Spring development of a Chlamydomonas population in Lake Nimetön,a small humic forest lake in southern Finland

Biomass development and vertical distribution of a Chlamydomonas population in a small humic forest lake was followed by daily sampling in May-June, 1984. Chlamydomonas dominated the phytoplankton spring bloom, forming 71% of the maximum phytoplankton biomass on 18 May. In early May the outflow rate was high and during the 24 hour period when the maximum rate of surface runoff was recorded (8–9 May), 43% of the Chlamydomonas biomass was flushed out of the lake, which delayed the onset of biomass increase. When surface runoff had slowed down Chlamydomonas biomass started increasing and during wax of the population most cells were < 10 µm in diameter. Population maximum lasted for one day (18 May) and there-after Chlamydomonas biomass decreased towards the end of the study. During wane of the population most cells were > 10 µm in diameter.     

The interaction of ethanol and exogenous arachidonic acid in the generation of extracellular messengers by mouse peritoneal macrophages

It is increasingly recognized that macrophages play a crucial role in the development of chronic inflammatory states such as alcoholic liver disease. These cells can metabolize free arachidonic acid in the absence of a discernible trigger. The present study was undertaken to examine the short-term effects of ethanol on the generation of these exogenous arachidonate-derived extracellular mediators. Ethanol caused a dose-dependent decrease in the production of both cyclooxygenase and lipoxygenase metabolites. Similar effects were observed on the esterification of exogenous arachidonate into cellular lipids. To characterize further the effects of ethanol on exogenous arachidonic acid metabolism, we studied the short-term responses displayed by macrophages challenged with another soluble stimulus; the tumor-promoting agent phorbol myristate acetate. We observed an inhibition by ethanol of the superoxide anion response triggered by phorbol myristate acetate similar to that observed for exogenous arachidonate oxygenation. Our results show that ethanol can inhibit these soluble stimuli-elicited responses, possibly through its disorganizing effect on plasma membrane.     

Correlation between low CSA plasma concentration and severity of acute GvHD in bone marrow transplantation

Between 1982 and 1986 51 patients were treated with ciclosporin a (CSA) to prevent graft versus host disease (GvHD) after bone marrow transplantation (BMT). Major side effects of the drug were tremor, hypertension, hepatotoxicity and nephrotoxicity. Acute GvHD 0 degree to II degree occurred in 80% of our patients, and GvHD III degree and IV degree in 20% despite the use of CSA. Two to four days before the onset of GvHD, CSA serum levels were significantly lower on the average in patients who developed GvHD III degree and IV degree compared to the others. Our data indicate that plasma CSA concentrations higher than 250 ng/ml should be achieved to reduce the severity of GvHD after BMT.     

Effect of glycosylation on the mechanism of renaturation of invertase from yeast

N-Glycosylation occurs cotranslationally as soon as the growing polypeptide chain enters the endoplasmic reticulum, before the final native-like folded state is reached. We examined the role of the carbohydrate chains in the mechanism of protein folding. The in vitro folding and association of yeast invertase are used as an experimental system. External invertase contains approximately 50% carbohydrate, whereas cytoplasmic invertase is not glycosylated. The functional native state of both proteins is a homodimer. At pH greater than or equal to 6.5 and at protein concentrations below 3 micrograms/ml, the kinetics of reactivation and the final yields are similar for the two invertases. For both proteins, reactivation is a sequential reaction with a lag phase at the beginning. The nonglycosylated protein tends to aggregate during reactivation at low pH and at protein concentrations above 3 micrograms/ml. After separation of inactive material, the renatured protein is indistinguishable from the original native state by a number of physicochemical and functional criteria. The results suggest that the carbohydrate moiety does not affect the mechanism of folding and association of invertase. However, glycosylation improves the solubility of unfolded or partially folded invertase molecules and hence leads to a suppression of irreversible aggregation. Such a protective effect may also be important for the in vivo maturation of nascent glycosylated protein chains.     

Independence of in vitro iron absorption from mucosal transferrin content in rat jejunal and ileal segments

Isolated non blood-perfused intestinal segments from normal and iron-deficient rats were used in vitro. A modification of the luminal perfusion method according to Fisher and Parsons allowed the comparison of iron and transferrin quantities in the serosal fluid at 15 min intervals. Iron transfer in jejunal and ileal segments was directly proportional to the luminal iron concentration within a dose range of 1 to 100 mumol/l, did not show saturation characteristics and was linear over time. Jejunal segments from iron-deficient rats transferred about twice as much iron as the jejunal controls. In ileal segments there was no difference in iron transfer between iron-deficient and control rats; in both cases transfer amounted to approx. 10% of jejunal controls. An exponential correlation was found, when the decreasing transferrin content of the tissue was plotted against the cumulative water transport. Transferrin and albumin release from jejunal and ileal segments into the absorbate cumulated asymptotically, which is typical for wash-out phenomena. As iron transfer cumulated linearly while transferrin release cumulated in an asymptotic manner, the capacity of transferrin to bind iron ions is exceeded roughly 100 times by molar equivalents of iron in the last absorbate fractions. Independence of iron transfer from mucosal transferrin quantities is concluded. As the molar transferrin/albumin ratios do not show significant differences between plasma and the sequence of absorbate samples, a wash-out from the gut's interstitial space is assumed, which makes plasma the most likely origin of transferrin in the mucosa.