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The purpose of this study was to test the hypothesis that hypochromic reticulocytes, hypochromic erythrocytes and p-transferrin receptors are sensitive variables in detecting iron-deficient erythropoiesis in healthy subjects and hemodialysis patients.Study 1: Twenty-one blood donors donated 450 mL blood. During the following 2 weeks blood samples were analyzed for the variables mentioned above. Study 2: Twenty-eight blood donors received 10,000 U recombinant human erythropoietin (rHuEPO) twice in the first week or placebo, after they had donated 450 mL blood. During the following 3 weeks the blood samples were analyzed for the variables mentioned in Study 1. Study 3: Eighteen hemodialysis patients receiving rHuEPO and iron treatment had either iron treatment discontinued for 4 weeks, after which iron was resumed, or received unchanged treatment. During 8 weeks blood samples were analyzed for the variables mentioned in Study 1.Study 1: Blood donation induced an increase in hypochromic reticulocytes of 178%, in hypochromic erythrocytes the increase was 267%, and in p-transferrin receptors 32%. Study 2: Treatment with rHuEPO induced a more pronounced increase than placebo in hypochromic reticulocytes (232% vs. 158%) and hypochromic erythrocytes (1240% vs. 300%), but not in p-transferrin receptors. Study 3: Discontinuation of iron treatment did not cause any significant differences in the variables mentioned above between the two groups, but caused a 25% decrease in p-ferritin. When iron treatment was resumed, p-ferritin increased by 19%. We found no significant changes in the control group.Hypochromic reticulocytes, hypochromic erythrocytes and p-transferrin receptors are sensitive variables in the early detection of iron-deficient erythropoiesis in healthy subjects, but in this study the iron withdrawal period was too short to show the value of these variables in the detection of iron-deficient erythropoiesis in hemodialysis patients.

The effect of enterocyte-like differentiation on the transferrin receptor (TfR) polarity in filter-grown Caco-2 cells was studied. The ratio of apical to basolateral TfRs which was found to be approximately 1:1 on the first day after the cells had reached confluence, changed to 1:40 eight days after reaching confluence. The transepithelial electrical resistance (TEER), transport of horseradish peroxidase (HRP) across the monolayer, and total cellular TfR number remained constant over this period. However, the activity of brush border membrane-associated alkaline phosphatase, an established marker for enterocyte differentiation, increased over this 8-day period concurrent with a decrease in apical TfR number. These results suggest that enterocyte-like differentiation rather than tight junction formation is most likely responsible for the polarized distribution of TfRs in Caco-2 cells. The effects of the fungal metabolite brefeldin A (BFA) on TfR distribution and TfR-mediated transcytosis in Caco-2 cells were also studied. BFA caused a marked decrease in the number of basolateral TfRs along with a slight increase in the number of apical TfR. BFA enhanced the TfR-mediated transcytosis of both 125I-Tf and the horseradish peroxidase-Tf conjugate across Caco-2 cells in both apical-to-basolateral and basolateral-to-apical directions. These findings imply a potential application of BFA as an enhancer for TfR-mediated delivery of protein drugs across the intestinal epithelium.

Indices of copper status, specifically serum copper and ceruloplasmin concentrations and erythrocyte superoxide dismutase activity, and iron status, including serum ferritin, transferrin receptors, hemoglobin and hematocrit, were studied in 27 college-aged females with adequate iron versus low iron stores.Serum copper and ceruloplasmin concentrations, erythrocyte superoxide dismutase activity, serum ferritin, transferrin receptors, hemoglobin and hematocrit were studied in 15 females with non-anemic iron depletion before and after five weeks of iron supplementation and in 12 healthy iron-adequate females aged 19 to 28 years.Mean hemoglobin, hematocrit and ferritin concentrations of the control group (144 +/- 11 g/L, 43 +/- 3% and 38 +/- 15 micro g/L, respectively) were significantly higher than those of the iron depleted group prior to supplementation (134 +/- 9 g/L, 39 +/- 2% and 11 +/- 6 micro g/L, respectively). The serum transferrin receptor to serum ferritin ratio was significantly greater for the iron depleted group prior to supplementation (890 +/- 753) versus the control group (151 +/- 61). Mean serum copper and ceruloplasmin concentrations and erythrocyte superoxide dismutase activity of the iron-adequate control group (20.0 +/- 5.7 micro mol/L, 463 +/- 142 mg/L and 527 +/- 124 U/mL, respectively) were significantly higher than those of the iron depleted group (12.4 +/- 3.8 micro mol/L, 350 +/- 108 mg/L and 353 +/- 186 U/mL, respectively) prior to supplementation. Following iron supplementation, hematocrit and ferritin concentrations of the iron depleted group significantly increased to 42 +/- 3% and 26 +/- 8 micro g/L, respectively. Mean serum transferrin receptor concentrations and the serum transferrin receptor to ferritin ratios significantly decreased in the iron depleted group following supplementation (6.1 +/- 1.6 mg/L to 4.6 +/- 1.5 mg/L and 890 +/- 753 to 198 +/- 114, respectively). Iron supplementation also significantly increased the mean serum copper concentration to 14.2 +/- 5.4 micro mol/L and, in subjects with serum ferritin concentrations/=12 micro g/L, the mean serum ceruloplasmin concentration.Non-anemic iron depletion characterized by low iron stores is associated with negative impacts on copper status. Iron supplements improved indices of iron status and serum copper and ceruloplasmin concentrations. Whether the diminished serum copper and ceruloplasmin concentrations and superoxide dismutase activity are associated with free radical damage to iron depleted cells requires further investigation.

There are few reports revealing association between iron intake and environmental lead exposure during pregnancy. Therefore, the present study was undertaken to investigate the effect of iron supplementation on biochemical modulation of certain lead toxicity markers associated with pregnancy. Iron and folic acid supplementations were given to 250 pregnant anemic women (mild = 100, moderate = 100 and severe = 50) and 100 age matched nonanemic pregnant women as controls for 100 days. Lead (Pb) toxicity markers, enzymatic and nonenzymatic antioxidant were estimated as per standard protocols. The levels of Pb, serum transferrin receptors (sTfR), zinc protoporphyrin (ZPP), δ-aminolevulinic acid (δ-ALA, both in blood and urine) were found significantly increased in all pretreated subjects and these were decreased after oral iron supplementation. Iron-deficient pregnant women reflected a significant increase in lipid peroxide levels (LPO) and protein carbonyl levels (PC) which were found to be further increased after iron supplementation. The levels of iron (Fe), haemoglobin (Hb), ferritin, delta aminolevulinic acid dehydratase (δ-ALAD), catalase (CAT), superoxide dismutase (SOD), and reduced glutathione levels (GSH) were significantly decreased in pretreated groups and these parameters were found significantly increased in all supplemented subjects after treatment. Antioxidant vitamins

Propose The early diagnosis of hepatocellular carcinoma (HCC) with ferritin heavy chain (Fth) modified by alpha-fetoprotein (AFP) promoter has been studied. However, no study has focused on the considerable upregulation and specific targeting effects of transferrin receptors (TfR) caused by the transfection of plasmids encoded with the AFP promoter. Thus, the objective of our study was to investigate whether the transfection of Fth gene modified with AFP promoter (AFP@Fth) could be used for early diagnosis and enhanced treatment of HCC. Methods The AFP@Fth plasmid was transfected into AFP positive cells. The expression of intracellular Ferritin was verified by Western blot, and the upregulation of TfR was confirmed by immunofluorescence and flow cytometry analysis. Cellular iron accumulation resulting in decreased imaging signals was examined by magnetic resonance imagining. Doxorubicin liposome modified with transferrin (Tf-LPD) was prepared to investigate the efficiency of the subsequent treatment after transfection. The enhanced drug distribution and effects were investigated both in vitro and in vivo. Results Both Ferritin and TfR were overexpressed after transfection. The transfected cells showed higher intracellular iron accumulation and resulted in a lower MR T2-weighted imaging (T2WI) intensity, suggesting that the transfection of AFP@Fth could be a potential strategy for early diagnosis of liver cancer. The following treatment efficacy was revealed by Tf-LPD. As compared with un-transfected cells, transfected cells exhibited higher uptake of transferrin-modified liposomes (Tf-LP), which was due to the specific interaction between Tf and TfR overexpressed on the transfected cells. This is also the reason why Tf-LPD showed better in vitro and in vivo anticancer ability than doxorubicin loaded liposome (LPD). These results suggested that transfection of AFP@Fth could result in enhanced therapy of liver cancer. Conclusion Transfection of AFP@Fth could be used for early diagnosis and for enhanced treatment of live cancers.

Glioblastomas (GBMs) are known to harbour subsets of cells known as tumour-initiating cells (TICs), which are responsible for the maintenance, invasiveness and recurrence of GBMs. Conventional chemotherapeutics act on rapidly dividing cells, sparing the TICs and result in tumour relapse. Resveratrol (RES) has shown chemopreventive effects in all the major stages of cancer including initiation, promotion and progression, but poor physicochemical and pharmacokinetic properties limit its use as a free drug. Hence we developed a liposomal formulation of RES (RES-L) to eradicate both the bulk tumour cells and TICs in GBMs. Since both these subpopulations of cells are known to over-express transferrin receptors, we developed transferrin-targeted RES-L (Tf-RES-L) to enhance tumour-specific delivery. We studied the effects of RES on neurospheres (NS) used as an in vitro model to study TICs derived from GBM cell lines. Free RES and RES formulations inhibited the anchorage-independent growth of GBM neurospheres. The NS-derived cells expressed TfRs and the Tf-targeted liposomes showed a significantly higher association with NS versus the non-targeted liposomes. Finally an increased activation of caspases 3/7 was seen when NS were treated with RES formulations. Together, these studies advocate for further investigations with RES-L and the use Tf to target the TIC populations.

Therapeutic outcome for the treatment of glioma was often limited due to the two barriers involved: the blood-brain barrier (BBB) and blood-brain tumor barrier (BBTB). Therefore, the development of nanocarriers that possess both BBB and BBTB permeability and glioma-targeting ability is of great importance for the chemotherapy of glioma. New frontiers in nanomedicine are advancing the research of new biomaterials. Here we constructed a natural high-density lipoprotein particle (HDL)-based drug delivery system with the dual-modification of T7 and dA7R peptide ligand (T7/dA7R-HDL) to achieve the above goals. HDL, the smallest lipoprotein, plays a biological role and is highly suitable as a platform for delivering imaging and therapeutic agents. T7 is a seven-peptide ligand of transferrin receptors (TfR) capable of circumventing the BBB and then targeting glioma. dA7R is a d-peptide ligand of vascular endothelial growth factor receptor 2 (VEGFR 2) overexpressed on angiogenesis, presenting excellent glioma-homing property. 10-Hydroxycamptothecin (HCPT), a hydrophobic anti-cancer drug, was used as the model drug in this study. By combining the dual-targeting delivery effect, the dual-modified HDL displayed higher glioma localization than that of single ligand-modified HDL or free HCPT. After loading with HCPT, T7/dA7R-HDL showed the most favorable anti-glioma effect in vivo. These results demonstrated that the dual-targeting natural nanocarriers strategy provides a potential method for improving brain drug delivery and anti-glioma treatment efficacy.

Off-target effects of drugs severely limit cancer therapy. Targeted nanocarriers are promising to enhance the delivery of therapeutics to tumors. Among many approaches for active tumor-targeting, arginine-rich cell penetrating peptides (AR-CPP) and ligands specific to target over-expressed receptors on cancer-cell surfaces, are popular. Earlier, we showed that the attachment of an AR-CPP octaarginine (R8) to the surface of DOXIL® (Doxorubicin encapsulated PEGylated liposomes) improved cytoplasmic and nuclear DOX delivery that enhanced the cytotoxic effect in vitro and improved therapeutic efficacy in vivo. Here, we report on DOX-loaded liposomes, surface-modified with, R8 and transferrin (Tf) (Dual DOX-L), to improve targeting of A2780 ovarian carcinoma cells via the over-expressed transferrin receptors (TfRs) with R8-mediated intracellular DOX delivery. Flow cytometry analysis with fluorescently labeled DualL (without DOX) showed two-fold higher cancer-cell association than other treatments after 4 h treatment. Blocking entry pathways of R8 (macropinocytosis) and Tf (receptor-mediated endocytosis, RME) resulted in a decreased cancer-cell association of DualL. Confocal microscopy confirmed involvement of both entry pathways and cytoplasmic liposome accumulation with nuclear DOX delivery for Dual DOX-L. Dual DOX-L exhibited enhanced cytotoxicity in vitro and was most effective in controlling tumor growth in vivo in an A2780 ovarian xenograft model compared to other treatments. A pilot biodistribution study showed improved DOX accumulation in tumors after Dual DOX-L treatment. All results collectively presented a clear advantage of the R8 and Tf combination to elevate the therapeutic potential of DOX-L by exploiting TfR over-expression imparting specificity followed by endosomal escape and intracellular delivery via R8.

Some anti-murine transferrin receptor monoclonal antibodies block iron uptake in mouse cell lines and inhibit cell growth. We report here the selection and characterization of mutant murine lymphoma cell lines which escape this growth inhibition by anti-transferrin receptor antibody. Growth assays and immunoprecipitation of transferrin receptor in hybrids between independently derived mutants or between mutants and antibody-susceptible parental cell lines indicate that all of the selected lines have a similar genetic alteration that is codominantly expressed in hybrids. Anti-transferrin receptor antibodies and transferrin itself still bind to the mutant lines with saturating levels and Kd values very similar to those of the parental lines. However, reciprocal clearing experiments by immunoprecipitation and reciprocal blocking of binding to the cell surface with two anti-transferrin receptor antibodies indicate that the mutant lines have altered a fraction of their transferrin receptors such that the growth-inhibiting antibody no longer binds, whereas another portion of their transferrin receptors is similar to those of the parental lines and binds both antibodies. These results argue that the antibody-selected mutant cell lines are heterozygous in transferrin receptor expression, probably with a mutation in one of the transferrin receptor structural genes.

Iron overload cardiomyopathy is one of the most common causes of death in thalassemia patients. Although new iron chelating agents have been developed, the mortality rate from heart failure remains elevated as a result of iron overload. This could be due to the fact that our understanding of the underlying mechanism of iron uptake into cardiomyocytes is still unclear, thus impeding the discovery and refinement of more effective therapy for thalassemia therapeutic strategies in thalassemic iron overload cardiomyopathy. Growing evidence indicates that multiple routes of iron entry into cardiomyocytes exist under iron overload conditions. These include both L-type (LTCC) and T-type (TTCC) calcium channels, divalent metal transporter 1 (DMT1) and transferrin receptors (TfRs). In this review, the routes of iron uptake into cardiomyocytes under iron overload conditions are presented. Evidence from pharmacological interventions in support or against the possible route of iron entry of each portal in cardiomyocytes are also comprehensively summarized and discussed.

Transferrin is one of acute phase reactants in inflamed lesion. Expression of transferrin receptor is increased in activated macrophage during inflammation process. Conjugates of target ligand with novel water-soluble chitosan for fast excretion of background radioactivity have been shown to be selectively taken up via the target molecules. In this study, radiolabeled 99mTc–Transferrin conjugate was synthesized and evaluated its efficacy in vivo as a targeted agent for the rapid detection of inflamed lesion that expresses relatively high level of transferrin receptors. Transferrin was conjugated with HYNIC-chitosan and radiolabeled with 99mTc. The biodistribution and scintigraphic images with the 99mTc–HYNIC–chitosan–Transferrin conjugate (99mTc–Tfc) were studied in a murine infection model in which the infection was induced with Escherichia coli (2 × 106 colonies). The %ID/g was as follows: 1.612 ± 0.098, 2.473 ± 0.202 and 2.617 ± 0.646% at 30, 120 min and 6 h after injection, respectively. Gamma camera ...

The high level of expression of transferrin receptors (Tf-R) on the surface of endothelial cells of the blood-brain-barrier (BBB) had been widely utilized to deliver drugs to the brain. The primary aim of this study was to use transferrin receptor mediated endocytosis as a pathway for the rational development of holo-transferrin coupled liposomes for drug targeting to the brain. Citicoline is a neuroprotective agent used clinically to treat for instance Parkinson disease, stroke, Alzheimer's disease and brain ischemia. Citicoline does not readily cross the BBB because of its strong polar nature. Hence, citicoline was used as a model drug. (Citicoline liposomes have been prepared using dipalmitoylphosphatidylcholine (DPPC) or distearoylphosphatidylcholine (DSPC) by dry lipid film hydration-extrusion method). The effect of the use of liposomes composed of DPPC or DSPC on their citicoline encapsulation efficiency and their stability in vitro were studied. Transferrin was coupled to liposomes by a technique which involves the prevention of scavenging diferric iron atoms of transferrin. The coupling efficiency of transferrin to the liposomes was studied. In vitro evaluation of transferrin-coupled liposomes was performed for their radioprotective effect in radiation treated cell cultures. In this study, OVCAR-3 cells were used as a model cell type over-expressing the Tf-R and human umbilical vein endothelial cells (HUVEC) as BBB endothelial cell model. The average diameter of DPPC and DSPC liposomes were 138 +/- 6.3 and 79.0 +/- 3.2 nm, respectively. The citicoline encapsulation capacity of DPPC and DSPC liposomes was 81.8 +/- 12.8 and 54.9 +/- 0.04 microg/micromol of phospholipid, respectively. Liposomes prepared from DSPC showed relatively better stability than DPPC liposomes at 37 degrees C and in the presence of serum. Hence, DSPC liposomes were used for transferrin coupling and an average of 46-55 molecules of transferrin were present per liposome. Free citicoline has shown radioprotective effect at higher doses tested. Interestingly, encapsulation of citicoline in pegylated liposomes significantly improved the radioprotective effect by 4-fold compared to free citicoline in OVCAR-3 but not in HUVEC. Further, citicoline encapsulation in transferrin-coupled liposomes has significantly improved the radioprotective effect by approximately 8-fold in OVCAR-3 and 2-fold in HUVEC cells with respect to the free drug. This is likely due to the entry of citicoline into cells via transferrin receptor mediated endocytosis. In conclusion, our results suggest that low concentrations of citicoline encapsulated in transferrin-coupled liposomes could offer therapeutic benefit in treating stroke compared to free citicoline.

Increasing evidence supports a role of cellular iron in the initiation and development of atherosclerosis. We and others reported earlier that iron-laden macrophages are associated with LDL oxidation, angiogenesis, nitric oxide production and apoptosis in atherosclerotic processes. Here we have further studied perturbed iron metabolism in macrophages, their interaction with lipoproteins and the origin of iron accumulation in human atheroma. In both early and advanced human atheroma lesions, hemoglobin and ferritin accumulation correlated with the macrophage-rich areas. Iron uptake into macrophages, via transferrin receptors or scavenger receptor-mediated erythrophagocytosis, increased cellular iron and accelerated ferritin synthesis at both mRNA and protein levels. The binding activity of iron regulatory proteins was enhanced by desferrioxamine (DFO) and decreased by hemin and iron compounds. Iron-laden macrophages exocytosed both iron and ferritin into the culture medium. Exposure to oxidized low-density lipoprotein (oxLDL,or=50 microg/mL) resulted in20% apoptosis of iron-laden human macrophages, but cells remained impermeable after a 24 h period and an increased excretion of ferritin could be observed by immunostaining techniques. Exposure to high-density lipoprotein (HDL) significantly decreased ferritin excretion from these cells. We conclude: (i) erythrophagocytosis and hemoglobin catabolism by macrophages contribute to ferritin accumulation in human atherosclerotic lesions and; (ii) iron uptake into macrophages leads to increased synthesis and secretion of ferritin; (iii) oxidized LDL and HDL have different effects on these processes.

Background: Anaemia and iron-deficiency anaemia in women are global problems that are prevalent throughout the reproductive cycle. Data are scarce on whether iron supplementation in pregnancy and puerperium has a sustained effect on haemoglobin concentration. Objective: To assess whether there is a dose effect of iron supplementation in pregnancy and puerperium on haemoglobin 6 weeks after delivery, and compare the effectiveness of daily and weekly dose regimens at 6 weeks postpartum. Design: 50 antenatal centres were assigned randomly to 1×60 mg iron daily or 2×60 mg once weekly. Data are reported for 146 women (daily, n=67; weekly, n=79): haemoglobin, serum ferritin (sFt) and serum transferrin receptors (sTfR) at baseline and at 6 weeks postpartum. Tablet intake was monitored using pill-bottles equipped with electronic counting devices. Results: There was a dose effect of iron supplementation on haemoglobin concentration at 6 weeks postpartum. Endpoint attaine d haemoglobin, sFt and sTfR did not differ between daily and weekly groups, although a larger increment of sFt was found in the daily group (p=0.03). Conclusions: Effects of iron supplementation in pregnancy and puerperium were observed at 6 weeks after delivery. The size of the effect was dependent on the number of tablets, not on daily or weekly regimen. It is not known whether the effects of iron supplementation in pregnancy are sustained into the next pregnancy. Keywords: Bangladesh, haemoglobin, iron status, iron supplementation, pregnancy, puerperium.

We investigated the expression of HOXB cluster genes in purified phytohemagglutinin (PHA)-activated T lymphocytes from normal adult peripheral blood by reverse transcription PCR and RNase protection. These genes are not expressed in quiescent T cells, except for barely detectable B1 RNA. After the PHA stimulus, HOXB gene activation initiates coordinately as a rapid induction wave in the 3'-->5' cluster direction (i.e., from HOXB1 through B9 genes). Thus, (i) expression of the foremost 3'-located B1 and B2 genes peaks 10 min after PHA addition and then rapidly declines, (ii) activation of B3, B4, and B5 begins 10 min after PHA addition and peaks at later times (i.e., at 120 min for B5), (iii) B6, B7, and B9 are expressed at a low level starting at later times (45 to 60 min), and (iv) B8 remains silent. Treatment of PHA-activated T lymphocytes with antisense oligonucleotides to B2 or B4 mRNA causes a drastic inhibition of T-cell proliferation and a decreased expression of T-cell activation markers (i.e., interleukin 2 and transferrin receptors). Similarly, treatment of CEM-CCRF, Peer, and SEZ627 T acute lymphocytic leukemia cell lines with anti-B4 oligomer markedly inhibits cell proliferation. Finally, T cells stimulated by a low dosage of PHA in the presence of 1 microM retinoic acid show a marked increase of both HOXB expression, particularly B2, and cell proliferation. These studies provide novel evidence on the role of HOX genes in adult cell proliferation. (i) Coordinate, early activation of HOXB genes from the 3'-->5' cluster side apparently underlies T-cell activation. (ii) The expression pattern in adult PHA-activated T cells is strikingly similar to that observed in retinoic acid-induced teratocarcinoma cells (A. Simeone, D. Acampora, L. Arcioni, P. W. Andres, E. Boncinelli, and F. Mavilio, Nature (London) 346:763-766, 1990), thus suggesting that molecular mechanisms underlying HOX gene expression in the earliest stages of development may also operate in activated adult T lymphocytes.

Newly identified iron (Fe)-binding proteins isolated from both rat and human duodenal mucosa permit a better understanding of Fe absorption. Mucins bind Fe at acid pH to keep it soluble and available for absorption at the more alkaline pH of the duodenum; this explains the development of Fe deficiency in achlorhydric subjects. Integrin was identified on the surface of enterocytes in association with radioiron and is believed to facilitate the transfer of Fe through the microvillous membrane. Mobilferrin, a 56 kDa Fe-binding protein, was identified in enterocyte cytosol. It coprecipitates with integrin and appears in close association with integrin in the apical cytoplasm of absorptive cells. We postulate it accepts dietary Fe from integrin and acts as the shuttle protein from Fe in the cytoplasm. Since Fe in enterocytes remains in equilibrium with body stores, we postulate mucosal Fe uptake is regulated by the number of Fe-binding sites either occupied or unoccupied by Fe on mobilferrin. Fe repletion of enterocytes from body stores is probably accomplished via transferrin receptors on the basal membranes of enterocytes. Increased transfer of Fe from blood into absorptive enterocytes occurs in Fe-replete animals to inhibit mucosal uptake of dietary Fe. Little transfer of Fe from plasma to enterocytes occurs in Fe deficiency. Enhanced mucosal transfer into the body occurs with increased body need for Fe. The exact mechanism for mucosal transfer of Fe into the plasma has not been defined but may also be mediated by an integrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Two single-chain immunotoxins directed at the human transferrin receptor have been constructed by using polymerase chain reaction-based methods. Anti-TFR(Fv)-PE40 is encoded by a gene fusion between the DNA sequence encoding the antigen-binding portion (Fv) of a monoclonal antibody directed at the human transferrin receptor and that encoding a 40,000-molecular-weight fragment of Pseudomonas exotoxin (PE40). The other fusion protein, DT388-anti-TFR(Fv), is encoded by a gene fusion between the DNA encoding a truncated form of diphtheria toxin and that encoding the antigen-binding portion of antibody to human transferrin receptor. These gene fusions were expressed in Escherichia coli, and fusion proteins were purified by conventional chromatography techniques to near homogeneity. In anti-TFR(Fv)-PE40, the antigen-binding portion is placed at the amino terminus of the toxin, while in DT388-anti-TFR(Fv), it is at the carboxyl end of the toxin. Both these single-chain immunotoxins kill cells bearing the human transferrin receptors. However, anti-TFR(Fv)-PE40 was usually more active than DT388-anti-TFR(Fv), and in some cases it was several-hundred-fold more active. Anti-TFR(Fv)-PE40 was also more active on cell lines than a conjugate made by chemically coupling the native antibody to PE40, and in some cases it was more than 100-fold more active.

We analysed subsets of tonsillar lymphocytes and activated cells in each subset by two-color flow cytometry. There were many helper T cells (CD 4+ leu 8-) and few inducer T cells (CD 4+ leu 8+) in the tonsil. The situation was the reverse in the peripheral blood. The tonsil had few suppressor T cells (CD 8+ CD 11+). The proportion of activated cells (HLA-DR+) was low in the peripheral blood and high in the tonsil. In the tonsil, the ratio of cells with transferrin receptors to total lymphocytes was higher in the B than in the T subset, and higher in the subset of CD 4+ than in that of CD 8+ cells. Activated and proliferating lymphocytes were more abundant in the tonsils of children than in those of adults and more abundant in patients with hyperplastic tonsillitis than in those with recurrent tonsillitis.