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Introduction of new myeloma therapies offers new options for patients refractory to immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs). In this multicenter study, patients with relapsed multiple myeloma, who have received at least three prior lines of therapy, are refractory to both an IMiD (lenalidomide or pomalidomide) and a PI (bortezomib or carfilzomib), and have been exposed to an alkylating agent were identified. The time patients met the above criteria was defined as time zero (T0). Five hundred and forty-three patients diagnosed between 2006 and 2014 were enrolled in this study. Median age at T0was 62 years (range 31–87); 61% were males. The median duration between diagnosis and T0was 3.1 years. The median number of lines of therapy before T0was 4 (range 3–13). The median overall survival (OS) from T0for the entire cohort was 13 (95% confidence interval (CI) 11, 15) months. At least one regimen recorded after T0in 462 (85%) patients, with a median (95% CI) progression-free survival and OS from T0of 5 (4, 6), and 15.2 (13, 17) months, respectively. The study provides the expected outcome of relapsed multiple myeloma that is refractory to a PI and an IMiD, a benchmark for comparison of new therapies being evaluated.

Infantile myofibroma (IM) is a benign mesenchymal disorder. Although it is the most common tumor of infancy, it is a relatively rare pathological entity. IM occurs predominantly before the age of two. It is characterized by tumor formation in skin, muscle, viscera, bone and subcutaneous tissues. The tumor can occur in either solitary or multicentric forms. The solitary form without visceral involvement usually has a benign course. Male gender predominates in the solitary form.

A study on the load carrying capacity of a sheathed parallel timber beam structure taking into account load sharing between timber beams is presented. Monte Carlo simulations are used to generate systems and to evaluate the influence of different parameters on the system behaviour. The variability in bending strength within and between timber elements is accounted for using a model by Isaksson (1999). A tri-linear load deformation relationship is used giving the beam a reduced load carrying capacity after initial failure. Experiments indicate that system failure can be defined to occur when two beams next to each other or any three beams fail. The properties of the sheathing are assumed to be deterministic and the stiffness of the joint between beam and sheathing is varied. The simulations give the load capacity for the weakest beam in the system (with no load sharing and no capacity of the beam after initial failure) and for the system failure. The results show a 19-30 % increase in load capacity for the full model with load sharing compared to a system where the weakest beam with no load sharing defines the load capacity. The failure mode considered is bending failure in the solid timber beam.

Background: Tissue factor (TF) is the main initiator of blood coagulation in vivo. Its increased expression on activated monocytes is associated with thrombotic complications and mortality in conditions such as sepsis, disseminated intravascular coagulation and coronary artery disease. Objective: The effect of the vitamin B derivative nicotinamide on endotoxin-induced monocyte TF and CD11b expression, soluble interleukin(IL)-6, and clotting onset time (COT) was studied. Methods: Experiments were conducted in human peripheral blood leukocyte suspensions and in whole blood from eight healthy volunteers. Free oscillating rheometry (measuring COT) and flow cytometry were applied to evaluate the effect of endotoxin on TF, CD11b, IL-6 and the overall coagulation response of plasma supplemented with activated autologous leukocytes. Results: In response to endotoxin, there was an increase in IL-6, TF and CD11b expression and a procoagulant shift of COT. At 4 mmol L−1nicotinamide, inhibition of TF expression and IL-6 and a normalization of COT were seen. At 16 mmol L−1nicotinamide, CD11b decreased also. The level of monocyte TF expression correlated with the COT readings, and the endotoxin-induced procoagulant shift of COT could be totally inhibited by blocking TF with an inhibitory antibody. Conclusions: These results demonstrate the ability of nicotinamide to inhibit the activation of coagulation associated with endotoxemia. We have previously shown that nicotinamide exerts strong anti-inflammatory effects. Evidence is accumulating for nicotinamide to have a therapeutic potential in modulating disease states in which there is a profound activation of coagulation and inflammation, such as in sepsis and disseminated intravascular coagulation.

For structural timber systems such as trusses, system effects related to strength variation within timber members has a significant effect on reliability. The system effect originates from the reduced probability that weak sections of timber coincide with the most stressed sections in the truss. In this paper, the strength variation within and between timber members is described with a statistical model earlier calibrated against data for Norway Spruce of Scandinavian origin. A method is presented to account for buckling effects when expressing the interaction between axial loading and bending in all sections of the truss members. The interaction is expressed in terms of a combined stress index, CSI, defined so that the failure criterion is CSI=1. For a given truss with given load, the cumulative distribution of CSI can be determined by Monte Carlo simulations based on the statistical model. By comparing the results with corresponding results obtained from deterministic engineering design, the statistical system effect is quantified. It is found that within member variability gives an extra capacity of 12% for Norway Spruce and 24% for Radiata Pine. Design rules proposed for Eurocode accounting for system effects in trusses give results slightly on the safe side compared to the results from the present study.

To study quality of life among patients living with a hereditary tumor syndrome, the small group with multiple endocrine neoplasia type 1 (MEN1) was selected. It is characterized by multifocal adenomas of the pancreas, parathyroid, anterior pituitary and other endocrine glands. Patients were assessed at an in-hospital stay and six months later at home. Patients at a specialist ward for MEN1 were recruited consecutively (n= 36) during one year. Eighty-one percent participated (n= 29). Four questionnaires were used: the Hospital Anxiety and Depression Scale (HADS), the Impact of Event Scale (IES), the Life Orientation Test (LOT) and the Short Form-36 (SF-36). Psychosocial outcome measures (anxiety, depression, intrusion, avoidance) changed only marginally between the in hospital stay and six months later at home. However, depression increased for patients categorized as having a high burden of disease and treatment. Compared to population-based norm values, the SF-36 scores of the patient group MEN1were lower for General Health and Social Functioning. Optimism assessed at the hospital was a predictor of Mental Health six months later. Most MEN 1 patients (70%) were pessimists. Patients having a higher burden of disease and treatment are in need of support after discharge. Patients could easily be monitored with questionnaires and, when indicated, offered help for their psychosocial distress.

In chlorophyll biosynthesis, insertion of Mg2+ into protoporphyrin IX is catalysed in an ATP-dependent reaction by a three-subunit (BchI, BchD and BchH) enzyme magnesium chelatase. In this work we present the three-dimensional structure of the ATP-binding subunit BchI. The structure has been solved by the multiple wavelength anomalous dispersion method and refined at 2.1 A˚ resolution to the crystallographic R-factor of 22.2 % (Rfree = 24.5 %). It belongs to the chaperone-like “ATPase associated with a variety of cellular activities” (AAA) family of ATPases, with a novel arrangement of domains: the C-terminal helical domain is located behind the nucleotide-binding site, while in other known AAA module structures it is located on the top. Examination by electron microscopy of BchI solutions in the presence of ATP demonstrated that BchI, like other AAA proteins, forms oligomeric ring structures. Analysis of the amino acid sequence of subunit BchD revealed an AAA module at the N-terminal portion of the sequence and an integrin I domain at the C terminus. An acidic, proline-rich region linking these two domains is suggested to contribute to the association of BchI and BchD by binding to a positively charged cleft at the surface of the nucleotide-binding domain of BchI. Analysis of the amino acid sequences of BchI and BchH revealed integrin I domain-binding sequence motifs. These are proposed to bind the integrin I domain of BchD during the functional cycle of magnesium chelatase, linking porphyrin metallation by BchH to ATP hydrolysis by BchI. An integrin I domain and an acidic and proline-rich region have been identified in subunit CobT of cobalt chelatase, clearly demonstrating its homology to BchD. These findings, for the first time, provide an insight into the subunit organisation of magnesium chelatase and the homologous colbalt chelatase.

Interleukin-2 is a remarkable activator of lymphocytes with anti-neoplastic properties such as T-cells or natural killer cells, but tumor regression only rarely occurs in interleukin-2-treated cancer patients. In this review, we focus on interactions between monocytes/macrophages and T-cells/natural killer-cells, and in particular the role of such interactions for the outcome of cancer immunotherapy with interleukin-2. We propose that interleukin-2 therapy should be supplemented with compounds that alleviate toxicity inflicted by monocyte/macrophagederived reactive oxygen metabolites within and around tumors. The hypothesis is founded on data demonstrating that (i) functions of intratumoral lymphocytes in many human malignant tumors are inhibited by reactive oxygen metabolites, generated by neighboring monocytes/macrophages, (ii) interleukin-2 only weakly activates T-cells or natural killer cells in an environment of oxidative stress, and (iii) inhibitors of the formation of reactive oxygen metabolites or scavengers of reactive oxygen metabolites, synergize with interleukin-2 to activate these lymphocyte subsets. We also review the preclinical background to the use of histamine dihydrochloride, an inhibitor of reactive oxygen metabolite formation in monocytes/macrophages, as a supplement to cancer immunotherapy with interleukin-2.

We report a method to generate dendritic cells (DC) from frozen leukapheresis products of patients with chronic myeloid leukemia (CML), using sterile culture bags and serum-free culture medium, ie conditions feasible for re-infusion into the patient as part of immunother-apeutic protocols. Leukapheresis products were stored from harvests performed either at diagnosis (13 patients) or after chemotherapy with subsequent granulocyte colony stimulating factor (G-CSF) administration (9 patients), for Peripheral Blood Stem Cell (PBSC) collections. In the presence of optimal concentrations of GM-CSF (50 ng/ml) and IL-4 (40 ng/ml) CML progenitors differentiated on day 7 and 14 of culture to DC, expressing CD1a, HLA-DRand CD86 surface antigens. Mature DCs exhibited on average 12-fold higher allo-stimulatory capacity for CD4+and CD8+cells compared to non-cultured PBMC in mixed lymphocyte reaction (MLR). Only DCs obtained from CML patients at diagnosis exhibited bcr/abl fusion gene when tested by fluorescentin situhybridization (FISH). CD34-selection on leukapheresis products from diagnosis (7 patients) resulted in later maturation of DCs (after 14–15 d), compared to the non-selected PBMC. CD34-selection significantly increased the DC growth, and improved the allo-stimulatory capacity in MLR (on average on day 14, 3.5- and 2.3-fold, respectively). Large differences were observed between individual patients and different leukapheresis products from the same patient. Our report demonstrates the possibility to generateex vivoautologous functionally active DC in CML in a way that allows their clinical application as immunotherapeutic agents.

Ferrochelatase, the enzyme catalyzing metallation of protoporphyrin IX at the terminal step of heme biosynthesis, was co-crystallized with an isomer mixture of the potent inhibitor N-methylmesoporphyrin (N-MeMP). The X-ray structure revealed the active site of the enzyme, to which only one of the isomers was bound, and for the first time allowed characterization of the mode of porphyrin macrocycle distortion by ferrochelatase. Crystallization of ferrochelatase and N-MeMP in the presence of Cu2+ leads to metallation and demethylation of N-MeMP. A mechanism of porphyrin distortion is proposed, which assumes that the enzyme holds pyrrole rings B, C and D in a vice-like grip and forces a 36 ° tilt on ring A.

Normal refraction of radar waves through the atmosphere prevents radar data at distant ranges from being quantitatively representative for the Earth's surface unless precipitation-related processes taking place between the radar echo and the surface are taken into account. A one-dimensional model is presented which uses a physical description of the processes involved in obtaining surface precipitation rate from a radar echo aloft, comprising local production, coalescence and evaporation. One or two cloud layers are assumed depending on the cloud base height. Input data are operationally analysed cloud base height, temperature, and humidity along with radar observations and their altitudes. At the moment, only precipitation as liquid water is assumed in the model. The method and results using it will be presented and discussed.

Rats were given radiotherapy (total dose 30 Gy) over the abdomen. Seven days later specimens of the duodenum were prepared for in vitroreceptor autoradiography using the radioligand [125I]VIP. The autoradiograms were quantitatively analyzed using a computer system. Histological examination revealed that a very marked reorganization of the mucosa had occurred in response to irradiation. Using receptor autoradiography, we found [125I]VIP-specific binding sites in the reorganizing mucosa, except where denudation had occurred. Such binding sites also occurred in the smooth muscle layer of the duodenal wall. The observations suggest that VIP has profound effects in radiation-induced enteropathy.

A total of 159 cases of chloracne reported in 1969––1975 in TCDD-contaminated production of the herbicide 2,4,5-T have been followed for mortality and morbidity up to 1996 when blood and urine tests were performed on 50 survivors of these exposed chemical workers and matched controls. In exposed, the most frequent cause of sick leave was chloracne which persisted in 32%. Neurological symptoms were reported frequently (44% sleep disturbance, 32% headache, 30% neuralgia). BSR, leucocytes, γ-GT, SGOT, and SGPT were significantly higher in exposed than in controls. The effects of exposure (P=0.002) and alcohol (P=0.002) on γ-GT were found to be independent of each other. Comparisons within the chloracne cohort showed significantly exposed TCDD per gram blood lipid in patients with a history of liver disease (mean 801 pg/g) than without (mean 407 pg/g). Other congeners were not found elevated but some higher chlorinated furans and PCBs were found reduced in patients with liver disease. In multiple regression analysis with the factors age, alcohol, and log TCDD, the effects of TCDD and its interaction with age were found significant, indicative of chronic liver damage after high TCDD exposure at a young age. The prevalence of neurological symptoms and signs of chronic liver disease were related to TCDD in blood and abnormal poryphyrins in urine. In 48% coproporphyrin I>III ratio was elevated, this group showing increased TCDD (mean 719 pg/g). These results contribute to the evidence that chloracne is not the only chronic disease which can be related to TCDD exposure, even 23 years after exposure and despite high intersubject variability of TCDD half-life and other exposures.

Abstract: The plasma sprya deposition of a zirconia thermal barrier coating (TBC) on a gas turbine component was examined using analytical and experimental techniques. The coating thickness was simulated by the use of commercial off-line software. The impinging jet was modeled by means of a finite difference elliptic code using a simplified turbulence model. Powder particle velocity, temperature history, and trajectory were calculated using a stochastic discrete particle model. The heat transfer and fluid flow model were then used to calculate transient coating and substrate temperatures using the finite element method. The predicted thickness, temperature, and velocity of the particles and the coating temperatures were compared with these measurements, and good correlations were obtained. The coating microstructure was evaluated by optical and scanning microscopy techniques. Special attention was paid to the crack structures within the top coating. Finally, the correlation between the modeled parameters and the deposit microstructure was studied.

An α/β barrel is predicted for the three-dimensional (3D) structure of Bacillus subtilis ferrochelatase. To arrive at this structure, the THREADER program was used to find possible homologous 3D structures and to predict the secondary structure for the ferrochelatase sequence. The secondary structure was fit by hand to the selected homologous 3D structure then the MODELLER program was used to predict the fold of ferrochelatase. Molecular biological information about the conserved residues of ferrochelatase was used as the criteria to help select the homologous 3D structure used to predict the fold of ferrochelatase. Based on the predicted structure possible, ligands binding to the iron and protoporphyrin IX are discussed. The structure has been deposited in the Brookhaven database as ID 1FJI. © 1997 Wiley-Liss Inc.

1All members of a Spanish family (father, mother and six children) developed chloracne.2The causative agent was found to be the family's stock of olive oil, which had become contaminated with polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), pentachlorophenol, and hexachlorobenzene.3The more highly chlorinated PCDDs, in particular octachlorodibenzo-p-dioxin, were the predominant congeners in the oil.4Three members of the family exhibited either an overt or a sub-clinical disturbance of kidney function. The father also had a chronic respiratory problem. These changes could not be unequivocally attributed to the PCDDs.5Experimental toxicity of the oil was limited to the development of an hepatic porphyria in mice.6A serum sample, taken 5 years after consumption of the oil ceased, contained high levels of the PCDDs and PCDFs. Extrapolation back to ingested dose was used to validate dosage estimates.7The use of toxicity equivalence factors (TEFs) provided estimates of cumulative dosage to produce chloracne as 0.13-0.31 μg 2378-TCDD kg-1(using EPA TEFs) or 6.7-16 μg 2378-TCDD kg-1(using Nordic/NATO TEFs).8This is the first incident in which human toxicity is related primarily to ingestion of PCDDs and for which estimates of dosage can be made.

Abstract:  The production of chlorine and vinyl chloride (VCM) is associated with the formation of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). The objective of the present study was to determine whether the levels and congener patterns of PCDDs and PCDFs in the blood of workers involved in the production of such substances could be related to their occupational environment. PCDD and PCDF levels in blood plasma from VCM and chloralkali workers and in referents were determined by means of high-resolution gas chromatography/high resolution mass spectrometry (HRGC/ HRMS) techniques and the results were evaluated through principal component analysis (PCA). The overall levels of PCDDs and PCDFs were low, but the relative congener distribution detected in the workers differed from that found in the referents. 1,2,3,4,6,7,8-HpCDF, 1,2,3,4,7,8- and 1,2,3,6,7,8-HxCDFs are congeners related to work in VCM and chloralkali industries. Exposure to PCDDs and PCDFs in these industrial environments changes the congener-distribution pattern in the blood of workers as compared with referents. A combination of isomer-specific analysis of PCDDs and PCDFs in blood plasma and PCA is suitable for the differentiation between nonoccupational and occupational exposure and provides a means of assessing workers’ exposure situation in different occupational settings.

Atrial natriuretic peptide has frequently been detected in the cardiac conduction system and has been shown to regulate some intracellular effects in Purkinje fibers. To determine if atrial natriuretic peptide works as an autocrine and/or paracrine hormone on cardiac Purkinje fibers, we examined the different parts of the conduction system in the bovine heart by use of in vitro receptor autoradiography. In no parts of the bovine conduction system were specific binding sites for [¹²⁵I] atrial natriuretic peptide observed, whereas the ventricular myocardium exhibited a large number of [¹²⁵I] atrial natriuretic peptide binding sites. This is the first morphologic study showing the presence of [¹²⁵I] atrial natriuretic peptide binding sites in the ventricular myocardium and their absence in the conduction system. The present observations together with results obtained in studies using other methods strongly suggest that natriuretic peptide receptors are localized on ventricular myocytes.

Familial hemophagocytic lymphohistiocytosis (FHL) is a frequently missed and almost uniformly fatal childhood disorder. It is characterized by fever, hepatosplenomegaly, cytopenia, coagulopathy, and hypertriglyceridemia. The pathogenesis of FHL is not known but the above clinical and laboratory findings are compatible with reported in vitro and in vivo effects of several inflammatory cytokines. We measured circulating interferon-gamma (IFN-gamma), tumor necrosis factor/cachectin (TNF), and interleukin-6 (IL-6) in nine children with FHL. During active disease, elevated IFN-gamma was detected in seven of seven children, TNF in six of six, and IL-6 in two of six children studied. Thus, important inflammatory cytokines are augmented in active FHL and may contribute to the pathogenesis of the disease. Soluble CD8 was also increased in seven of seven children, which suggests a pathophysiologic importance of cytotoxic T lymphocytes. Because FHL appears to be associated with a systemic hypercytokinemia, our results also indicate that studies of FHL may contribute to the understanding of cytokine effects in vivo. Moreover, FHL is a hereditary disorder, suggesting that the hypercytokinemia is caused by a genetic defect in cytokine regulation.

The effect of allogenic human natural killer (NK) cells on fresh leukemic cells from three patients was investigated. The low levels of leukemic target cell lysis in the conventional 51Cr-release assay contrasted with a pronounced inhibitory effect on the colony growth of the clonogeneic leukemic target cells (L-CFC). The ability of allogeneic lymphocytes to inhibit L-CFC increased if they were pretreated with interferon (IFN), which also increased their NK activity, monitored in parallel cytotoxicity assay, against K562. Furthermore, cell separation procedures, based on differences in density among nonadherent lymphocytes, revealed that only NK cell containing fractions were inhibitory. We have also compared the susceptibility to NK-mediated L-CFC inhibition of IFN pretreated leukemic target cells with that of nontreated target cells. As in the case of NK lysis in general, this pretreatment of target cells abolished the presumably NK-mediated L-CFC inhibition. In conclusion, these data provide the first indication that NK cells can inhibit the in vitro growth of fresh clonogenic leukemia cells from patients with nonlymphocytic leukemia. The identity of NK cells as effector is strongly suggested by Percoll separation and responsiveness to interferon; the final proof awaits more sophisticated purification of these cells.

We report in this study the distribution of10Be in the top 40 m of the Renland ice core (East Greenland) and in a 30 m long core from DML (Dronning Maud Land, Antarctica) for the period 1931–1988. The two sites show differences in10Be content, the Antarctica site showing smaller variance and a lower average10Be annual flux. Similarly, the average accumulation rate (cm water equivalent year−1) is higher in the Renland relative to DML. The variability in accumulation (precipitation) rates seems to explain part of the difference in10Be flux between the two polar sites. Cyclic fluctuations of10Be flux correlate with the 11-year sunspot number and cosmic ray intensity than with the aa index (perturbation of the geomagnetic activity by the solar wind). Our data corroborate10Be cyclic fluctuation pattern from the Dye 3 ice core and confirm a promising potential for correlation of global and local events.

An expression system to allow targeting of heterologous proteins to the cell surface of Staphylococcus xylosus, a coagulase-negative gram-positive bacterium, is described. The expression of recombinant gene fragments, fused between gene fragments encoding the signal peptide and the cell surface-binding regions of staphylococcal protein A, targets the resulting fusion proteins to the outer bacterial cell surface via the membrane-anchoring region and the highly charged cell wall-spanning region of staphylococcal protein A. The expression system was used to secrete fusion proteins containing sequences from a malaria blood-stage antigen and a streptococcal albumin-binding receptor to the cell surface of S. xylosus. Analysis of the recombinant cells by immunogold staining and immunofluorescence revealed that both the receptor and the malaria peptide were properly processed and exposed on the surface of the host cells. However, only approximately 40 to 50% of the recombinant cells were strongly stained with antiserum reactive with the albumin-binding receptor, while approximately 10 to 15% of the cells were stained with antiserum reactive with the malaria peptide. The incomplete staining of some of the cells suggests steric effects that make the recombinant fusion proteins inaccessible to the reactive antibodies because of variable cell wall structures. However, the results demonstrate for the first time that recombinant techniques can be used to express heterologous receptors and immunogens on the surface of gram-positive cells.

Summary Cell recognition and adhesion are important events in embryonic development as well as in adult physiology. In recent years several cell adhesion molecules (CAMs), that mediate adhesive interactions between vertebrate cells, have been identified and characterized. These CAMs are in general cell surface-associated high molecular weight glycoproteins. Two groups of CAMs have been classified: primary CAMs, that appear early in development; secondary CAMs, that become expressed later and with a more restricted tissue distribution. One example of a secondary CAM is cellCAM 105. This glycoprotein was originally identified in rat hepatocytes, and was shown to be involved in the reaggregation of freshly isolated hepatocytesin vitro. Physico-chemical studies on pure cellCAM 105 have demonstrated that it has adhesive properties and can bind to itself in a homophilic, calcium-independent reaction. Immunochemical and immunohistochemical investigations have shown that cellCAM 105 occurs in liver, several epithelia, vessel endothelia, platelets and polymorphonuclear leukocytes, and that it is expressed primarily in terminally differentiated cells or cells that are undergoing terminal differentiation. Available information suggests that cellCAM 105 has different functions in different cell types, and that the common functional denominator might be membrane-membrane binding. Recent data indicate that cellCAM 105 is a calmodulin-binding protein, suggesting that cellCAM-mediated cell binding could be involved in transmembrane signalling.

The Plasmodium falciparumclustered asparagine-rich protein (CARP) is a merozoite-associated antigen which contains approximately 30% asparagine. Analysis of the DNA sequences located 5′ of the cloned 1.4-kb CARP gene in the P. falciparumgenome suggests that this gene fragment may encode the complete CARP and that the gene product is a protein of Mr50,000. To analyze the immunogenicity of CARP, the gene was expressed as a fusion protein with staphylococcal protein A (SpA-CARP). Immunization of rabbits with SpA-CARP in Freund′s complete adjuvant (FCA) resulted in a strong antibody response against CARP as measured in ELISA. This response was efficiently boosted and sustained over a long time while that induced by two immunizations with SpA-CARP in ISCOMs was weak and of shorter duration. In both instances, the antibody levels against CARP were further increased by a second booster injection consisting of either SpA-CARP or CARP fused to the serum albumin-binding region (BB) of streptococcal protein G (BB-CARP) in PBS, indicating that immunizations with SpA-CARP in FCA or ISCOMs had induced a CARP-specific immunological memory. Boosting with BB-CARP in PBS was more efficient than boosting with SpA-CARP in PBS. In all rabbits, the antibodies obtained after the booster with CARP in PBS were the most efficient inhibitors of merozoite invasion in vitro. The antisera reacted with the intracellular parasite in immunofluorescence and with a band of Mr50,000 in immunoblotting while several high-molecular-weight components as well as the one of Mr50,000 were immunoprecipitated. The specificity of the antibody responses varied between the different rabbits as indicated in ELISA, with short synthetic peptides representing different CARP sequences. Taken together, the results suggest that a previously cloned genomic DNA fragment may encode the complete P. falciparumblood-stage antigen CARP and that CARP is immunogenic in rabbits both when administered in FCA or ISCOMs.

An α/β barrel is predicted for the three‐dimensional (3D) structure of Bacillus subtilisferrochelatase. To arrive at this structure, the THREADER program was used to find possible homologous 3D structures and to predict the secondary structure for the ferrochelatase sequence. The secondary structure was fit by hand to the selected homologous 3D structure then the MODELLER program was used to predict the fold of ferrochelatase. Molecular biological information about the conserved residues of ferrochelatase was used as the criteria to help select the homologous 3D structure used to predict the fold of ferrochelatase. Based on the predicted structure possible, ligands binding to the iron and protoporphyrin IX are discussed. The structure has been deposited in the Brookhaven database as ID 1FJI. © 1997 Wiley‐Liss Inc.

(First submitted 15 Nov 1991;accepted 11 December 1991)The effect of IFN-γ and TNF-α treatment of an ovarian carcinoma line on the sensitivity to lysis by specific CTL clones and non-specific Tumor Associated Lymphocytes (TAL), isolated from the ascites fluid, was analyzed. Thein vitroestablished TAL line displayed a non-specific lytic activity against the autologous tumor as well as against several allogeneic tumor lines. Pretreatment with IFN-γ alone, or in combination with TNF-α, rendered the carcinoma line less susceptible to lysis by the autologous TAL line. Conversely, susceptibility to lysis by tumor specific T cell clones, isolated from the TAL line, was increased as a result of cytokine pretreatment. Several TCR-α/β+, CD8+T-cell clones showing a more specific pattern of lysis against the autologous tumor were isolated. Lysis of the autologous tumor by these clones involved the TCR-α/β via a MHC-class I restricted mechanism dependent on the adhesion molecules ICAM-1 and LFA-3, as inferred from antibody blocking studies. The enhanced sensitivity to specific CTL clones seen after cytokine treatment may be related to theenhanced expression of ICAM-1 molecules on the ovarian carcinoma. These results have implications for cytokine based immunotherapy, where IFN-γ may enhance the effects of tumor associated specific CTL while decreasing that of non-specific effector cells.

Mutations that cause a block in a late step of the protoheme IX biosynthetic pathway, i.e., in a step after uroporphyrinogen III, map at 94 degrees on the Bacillus subtilis chromosomal genetic map. We have cloned and sequenced the hem genes at this location. The sequenced region contains six open reading frames: ponA, hemE, hemH, hemY, ORFA, and ORFB. The ponA gene product shows over 30% sequence identity to penicillin-binding proteins 1A of Escherichia coli, Streptococcus pneumoniae, and Streptococcus oralis and probably has a role in cell wall metabolism. The hemE gene was identified from amino acid sequence comparisons as encoding uroporphyrinogen III decarboxylase. The hemH gene was identified by enzyme activity analysis of the HemH protein expressed in E. coli. It encodes a water-soluble ferrochelatase which catalyzes the final step in protoheme IX synthesis, the insertion of ferrous iron into protoporphyrin IX. The function of the hemY gene product was not elucidated, but mutation analysis shows that it is required for a late step in protoheme IX synthesis. The hemY gene probably encodes an enzyme with coproporphyrinogen III oxidase or protoporphyrinogen IX oxidase activity or both of these activities. Inactivation of the ORFA and ORFB genes did not block protoheme IX synthesis. Preliminary evidence for a hemEHY mRNA was obtained, and a promoter region located in front of hemE was identified. From these combined results we conclude that the hemEHY gene cluster encodes enzymes for the synthesis of protoheme IX from uroporphyrinogen III and probably constitutes an operon.

In vitro infection of human B lymphocytes with Epstein-Barr virus (EBV) results in establishment of B lymphoblastoid cell lines that reflect normal B cell phenotypes. In this study we have investigated whether immature B cells from fetal bone marrow and liver can serve as targets for EBV. The fetal bone marrow cells were readily transformed by EBV. Among the resulting cell lines, five were surface Ig (sIg)-negative. Three B cell-associated antigens defined by monoclonal antibodies were expressed to the same extent on the fetal cell lines, whether they belonged to the sIg- or sIg+ group. The various differentiation stages that these cell lines may represent are discussed.

We have recently reported (M. Petricek, L. Rutberg, I. Schröder, and L. Hederstedt, J. Bacteriol. 172: 2250-2258, 1990) the cloning and sequence of a Bacillus subtilis chromosomal DNA fragment containing hemA proposed to encode the NAD(P)H-dependent glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid (ALA) synthesis, hemX encoding a hydrophobic protein of unknown function, and hemC encoding hydroxymethylbilane synthase. In the present communication, we report the sequences and identities of three additional hem genes located immediately downstreatm of hemC, namely, hemD encoding uroporphyrinogen III synthase, hemB encoding porphobilinogen synthase, and hemL encoding glutamate-1-semialdehyde 2,1-aminotransferase. The six genes are proposed to constitute a hem operon encoding enzymes required for the synthesis of uroporphyrinogen III from glutamyl-tRNA. hemA, hemB, hemC, and hemD have all been shown to be essential for heme synthesis. However, deletion of an internal 427-bp fragment of hemL did not create a growth requirement for ALA or heme, indicating that formation of ALA from glutamate-1-semialdehyde can occur spontaneously in vivo or that this reaction may also be catalyzed by other enzymes. An analysis of B. subtilis carrying integrated plasmids or deletions-substitutions in or downstream of hemL indicates that no further genes in heme synthesis are part of the proposed hem operon.

The question whether penicillin V (pcV) given intermittently upon signs of upper respiratory tract infections (URTI) in otitis-prone children might prevent recurrent bouts of acute purulent otitis media (AOM) is addressed. As compared with continuous long-term antibiotic treatment as prophylaxis in otitis-prone children, intermittent administration would reduce the overall consumption of antibiotics. Seventy-six otitis-prone children less than 18 months of age were included in this double-blind, randomized, placebo-controlled multicentre study. Follow-up was from January till June. One hundred and twenty-three episodes of AOM occurred. The number of AOM episodes was reduced by 50% in the children on pcV during URTI episodes as compared with those on placebo. No obvious ecological drawbacks were noted. Thus, the described mode of pcV administration seems to be a rational and safe way to reduce the number of AOM episodes in otitis-prone children.

We analyzed the chemical compositions of dust and sea‐salt particles in the EPICA Dome C (EDC) ice core during 26–7 kyr BP using an ice‐sublimation technique and compared the results with existing data of the Dome Fuji (DF) ice core. Combined with ion concentration data, our data suggested similar sea‐salt fluxes in both cores and significantly lower dust flux in the EDC core. The differences in modal size and aspect ratio of dust particles between the two cores support the dominance of Patagonian source suggested by earlier works. The compositions of calcic dust showed major change at ~17 kyr BP, possibly reflecting a relative increase in dust transported via the upper troposphere. The calcium sulfate fraction was higher in the DF core than in the EDC core after ~17 kyr BP, suggesting that higher Patagonian dust contribution to the DF region. Abundant NaCl particles were found in the DF core in comparison with the EDC core from the LGM to early Holocene, possibly because of the high concentration of terrestrial dust in the DF core that reduced acid availability for sea‐salt modification. During the Holocene, the lower NaCl fraction and Cl−/Na+ratio in the EDC core suggested that most Cl−was lost to the atmosphere from snow at Dome C, while it was preserved at Dome Fuji as NaCl and solid solution. Dust flux in the Dome C core is significantly lower than in the Dome Fuji core from the LGM to the early HoloceneComparison of dust size and aspect ratio between the two cores suggest dominance of Patagonian dust source during the LGMMost Cl was lost to the atmosphere from snow at Dome C, while it was preserved at Dome Fuji as NaCl and in solid solution in the Holocene

IMMUNE reactions of ‘conventional’ T and B lymphocytes are generally thought to constitute major parts of any measurable, ‘specific’ tumour resistance against autologous tumours. There is no doubt that protective immunity can be induced against subsequently transplanted syngeneic tumours in experimental systems1. But, there is only scanty evidence to suggest that conventional immune reactions can provide resistance against such tumour cells when transplantation is made into normal individuals. Yet, it is frequently found that even long-transplanted syngeneic tumour cells may require comparatively high numbers of cells to ensure tumour take in normal recipients. Mice failing to succumb to small numbers of tumour cells can frequently be shown to reject a second graft of the same tumour with similar vigour2. Thus, natural resistance against tumours may occur with no display of classical, immunological memory. That such natural protective forces do exist against tumours has been claimed for many years3, but its underlying basis is poorly understood. Here, we present data indicating that naturally occurring killer cells may play a decisive part in providing resistance against syngeneic tumour cells in vivo.