Severe hypochromic microcytic anemia caused by a congenital defect of the iron transport pathway in erythroid cells

Abstract

We read with interest the paper from Jilani et al 1 in which rituximab treatment appeared to down-modulate CD20 expression through a combination of internalization and RNA regulation. The result is unexpected because previous studies had shown that CD20 is not modulated by monoclonal antibody (mAb) treatment, 2-4 even in vivo. 5 The study by Jilani et al used an anti-mouse immunoglobulin polyclonal antibody (Ab) that binds to the mouse V regions in rituximab. This reagent will bind to rituximab, while the chimeric antibody is coated onto CD20 ϩ cells. We are concerned that the apparent loss of binding reported may be due to factors other than loss of CD20 expression-specifically blocking by normal immunoglobulin.As part of our ongoing CD20 studies, we have developed a mouse anti-idiotype (Id) mAb (2A4) specific for the V regions of rituximab and its parent Ab 2B8 (M.S.C. and M.C.B., manuscript submitted, March 2004). This reagent binds rituximab or 2B8 while it is coated onto CD20 ϩ cells ( Figure 1A). Using this highly specific reagent we can find no evidence that CD20 is down-modulated on malignant B cells in either the presence or absence of plasma ( Figure 1B). Using the anti-Id mAb (MB2A4), together with a human Fc␥-specific mAb, we examined the ability of rituximab and its murine counterpart 2B8 to bind to CD20 on Raji cells and fresh B-cell chronic lymphocytic leukemia (B-CLL) cells with and without plasma. In these experiments, cells were incubated for 2 hours at 37°C as described by Jilani et al, before the cells were washed twice by centrifugation and stained for surface CD20 mAb. Interestingly, when using fluorescein isothiocyanate (FITC)-conjugated anti-Fc␥ mAb, our ; and chLOB7-4/7-6 [CD40]), cells were washed twice in phosphate-buffered saline/bovine serum albumin (BSA)/Azide before being stained with FITC-labeled mAb MB2A4 (10 g/mL) for 15 minutes at room temperature and being analyzed by flow cytometry. As can be seen, binding of 2A4 was observed only on cells coated with rituximab or its parent mAb 2B8, demonstrating both the ability of the mAb to detect rituximab when it was bound to cells and its specificity for the rituximab V regions. (B) Typical dot-plots generated by staining B-CLL cells incubated with either rituximab or 2B8 in the presence or absence of 33% plasma for 2 hours at 37°C, followed by washing, as indicated in panel A, and staining with either FITC-labeled anti-Id (2A4) or FITC-labeled anti-human Fc␥ (Hu Fc␥, SB2H2) for 30 minutes at room temperature and analysis by flow cytometry. The anti-Id mAb was able to detect both mAbs equally well in the presence or absence of plasma, whereas the Hu Fc␥ mAb could not detect rituximab after incubation in the presence of plasma. (C) The importance of wash steps in the detection of rituximab. After binding and incubation as in panel B, B-CLL cells were washed 1, 2, or 3 times by standard centrifugation prior to staining with either FITC-labeled anti-Id (MB2A4) or FITC-labeled Hu Fc␥ as described in panel B. Flow cytometry data are expre...

Functional consequences of the human DMT1 (SLC11A2) mutation on protein expression and iron uptake

“…These data show that disrupted complex glycosylation of DEL DMT1 results in retention of the immature form of this protein in the ER. The observed quantitative DMT1 protein reduction in the patient's bone marrow erythroblasts 9 and in the patient's BFU-Es indicates that the stability of DEL DMT1 protein might be reduced. As only 10% of the total DMT1 transcript in the patient's erythroid cells encodes for a fully functional E399D protein, it is likely that the erythroid iron use is severely suppressed.…”

“…10 However, it is difficult to make definitive conclusions as both intra-individual and inter-individual differences in the level of DMT1 expression in the duodenum, reflecting the body iron stores and recent iron content in the diet, may exist. Nevertheless, we believe that the liver hemosiderosis evolved as a result of the alteration of a few processes, including activation of heme absorption 9 and increased activity of the basolateral iron transport. 10 …”

“…The ethics committee of the Palacky University Hospital approved the study. Peripheral blood was collected in heparinized tubes and processed for in vitro colony-forming assay 9 and for Western blot analyses. Red blood cells were removed by treatment with NH 4 Cl.…”

“…We previously reported a Czech female with severe hypochromic microcytic anemia and iron overload caused by a homozygous mutation in the DMT1 gene (1285G Ͼ C) that changes Glu 399 to Asp (E399D). 9,10 This single nucleotide substitution also causes preferential skipping of exon 12 during mRNA processing. As a consequence, there are 2 different DMT1 transcripts present in the patient's cells: a full-length transcript containing the point mutation and that missing exon 12; the later version comprising 90% of the total DMT1 mRNA.…”

The homozygous mutation G75R in the human SLC11A2 gene leads to microcytic anaemia and iron overload

“…This would be supported by the hypothesis that the defective function of the endosomal SLC11A2 in hepatocytes impairs the iron efflux to the cytosol, leading to iron accumulation in a compartment that does not trigger ferritin synthesis (Priwitzerova et al, Beaumont et al, 2006). Moreover, the intestinal absorption of haem iron compensates for deficient ferrous iron uptake (Priwitzerova et al, 2004;Beaumont et al, 2006;Iolascon et al, 2006). The volume of transfusions in this patient (approximately 1 g of iron) should not justify the moderate iron deposit in the liver.…”