J.B.M. Boezeman

Magnetic resonance tracking of dendritic cells in melanoma patients for monitoring of cellular therapy

The success of cellular therapies will depend in part on accurate delivery of cells to target organs. In dendritic cell therapy, in particular, delivery and subsequent migration of cells to regional lymph nodes is essential for effective stimulation of the immune system. We show here that in vivo magnetic resonance tracking of magnetically labeled cells is feasible in humans for detecting very low numbers of dendritic cells in conjunction with detailed anatomical information. Autologous dendritic cells were labeled with a clinical superparamagnetic iron oxide formulation or 111In-oxine and were co-injected intranodally in melanoma patients under ultrasound guidance. In contrast to scintigraphic imaging, magnetic resonance imaging (MRI) allowed assessment of the accuracy of dendritic cell delivery and of inter- and intra-nodal cell migration patterns. MRI cell tracking using iron oxides appears clinically safe and well suited to monitor cellular therapy in humans.

Haploinsufficiency of TNXB Is Associated with Hypermobility Type of Ehlers-Danlos Syndrome

To the Editor: Ehlers-Danlos syndrome (EDS) is a heterogeneous group of heritable connective-tissue disorders, generally affecting skin, joints, and blood vessels. The most recent classification recognizes six subtypes (Beighton et al. 1998), of which the hypermobility type (HT-EDS [formerly EDS type III] [MIM 130020]) is the most common. This type of EDS is similar to benign joint hypermobility syndrome (BJHS), and both are often considered to represent the same hyperlaxity syndrome, since no clear clinical distinction can be made (Grahame 1999). Although various causative genes have been found in all other types of EDS, the genetic basis of HT-EDS or BJHS remains unexplained (Steinmann et al. 2002). One family has been described that has a missense mutation in COL3A1 (Narcisi et al. 1994), resulting in a phenotype that resembles HT-EDS, without obvious vascular complications. Mutations in COL3A1 generally result in the severe vascular type of EDS (MIM 130050). To our knowledge, no other cases of COL3A1 mutations in HT-EDS have been reported. Recently, we showed that deficiency of the extracellular-matrix protein tenascin-X (TNX), encoded by the TNXB gene, causes a new type of recessively inherited EDS (Schalkwijk et al. 2001). Patients with complete deficiency of TNX showed marked joint hypermobility, skin hyperextensibility, and easy bruising. The absence of atrophic scars and recessive inheritance distinguishes TNX deficiency from the classical type of EDS. In our initial report (Schalkwijk et al. 2001), only a few heterozygous family members were available for examination. Here, we have examined all 20 heterozygous family members (individuals from families A–D in table 1) who were available for further study, regardless of clinical symptoms; in all of these individuals, we have found significantly reduced serum TNX levels (56% ± 6% vs. 100% ± 14% in the control population; P<.001, by Students t test) (fig. 1f), and, in 17 of them, we have confirmed heterozygosity for a truncating TNXB mutation (table 1). Clinical examination revealed generalized joint hypermobility in nine family members (45%), using the Beighton score (Beighton et al. 1973), for HT-EDS, or the Brighton criteria (Grahame et al. 2000), for BJHS (table 1 and fig. 1e). Skin hyperextensibility and easy bruising, frequently seen in the individuals with complete TNX deficiency, were absent. A number of patients with haploinsufficiency had recurring joint dislocations and chronic joint pain, as are seen in HT-EDS and BJHS. Only four family members carrying two normal TNXB alleles were available for study, of whom none had hypermobility. The local medical ethics committee (CMO Regio Arnhem-Nijmegen) approved the study protocol, and informed consent was obtained from all patients. Figure  1 TNXB haploinsufficiency and generalized joint hypermobility. a–d, Pedigrees of families A–D (also see table 1). e, Joint hypermobility in individual III9 from family A. f, Distribution of serum TNX levels in control population, population ... Table 1 Clinical and Molecular Findings in Individuals with TNXB Haploinsufficiency/Reduced Serum TNX Levels A striking finding is that 0 of the 6 males with haploinsufficiency fulfilled the clinical criteria for HT-EDS or BJHS, whereas 9 of 14 (64%) females were positive. This finding is in accordance with previous population-based studies that show a female preponderance in joint hypermobility syndromes (Larsson et al. 1987; Rikken-Bultman et al. 1997). In a control group of 30 unaffected females of the same age as the females with haploinsufficiency in the present study, we found no individuals with a Beighton score >4. This indicates that the prevalence of generalized joint hypermobility in a population of females with haploinsufficiency is significantly higher than in a control population (P<.001, by χ2 test). No sex differences in serum TNX levels in unaffected individuals and individuals with haploinsufficiency were found (not shown). Because our observations in families carrying previously described TNXB mutations suggested an association between TNXB haploinsufficiency and joint hypermobility, we wondered about the prevalence of TNXB haploinsufficiency in patients with HT-EDS. We measured serum TNX levels (by ELISA) in an unselected cohort of 80 patients with HT-EDS who were recruited through the Dutch organization for patients with EDS. All patients were diagnosed with HT-EDS by a medical specialist, and ∼90% were female. Although the mean serum TNX level was not different in the cohort with HT-EDS overall (99.4%±19.7%) (fig. 1f), six of these patients (7.5% [all female]) had serum TNX levels >2.5 SDs (65%) below the mean for unaffected individuals. On the basis of the normal distribution of serum TNX levels, only 0.6% of individuals would be expected to have such low serum TNX levels, which is significantly less than the frequency found in the population with HT-EDS described in the present study (P<.001, by Fishers exact test). Clinically, patients with reduced TNX levels showed hypermobile joints, often associated with joint subluxations and chronic musculoskeletal pain (table 1). The clinical findings in these patients differ from those with complete TNX deficiency. Patients with haploinsufficiency do not have skin hyperextensibility and lack the easy bruising seen in patients with TNX deficiency. In addition, TNXB haploinsufficiency is expected to be an autosomal dominant trait, which is in accordance with the observed mode of inheritance of HT-EDS and BJHS. On screening for the presence of a 30-kb deletion described previously (Burch et al. 1997; Schalkwijk et al. 2001), we found that this deletion was present in one of these six patients. The 30-kb deletion creates a fusion gene of TNXB and XA, a partial duplicate of TNXB. The XA gene has an internal deletion that truncates its ORF, rendering XA and the fusion gene nonfunctional (Gitelman et al. 1992). The deleted allele also lacks CYP21, so this individual is also a carrier for congenital adrenal hyperplasia. Subsequently, we PCR amplified and directly sequenced the coding regions and the intron-exon boundaries of TNXB in the other five patients presumed to have haploinsufficiency (for primers used, see Schalkwijk et al. 2001). One patient (individual F in table 1) was heterozygous for a 2-bp deletion, [AA56063] del, in exon 8, resulting in a premature stop codon at the position of amino acid 1231. In the other four patients, we were unable to identify mutations in TNXB. These patients may have mutations, in regulatory sequences or in exons of the TNXB gene, that have not yet been identified, or they may represent the extreme in normal variation of TNX expression. In conclusion, in the present study, we have reported a genetic defect associated with HT-EDS or BJHS. On the basis of the observed phenotype in patients with complete TNX deficiency and the high prevalence of generalized joint hypermobility in heterozygous females, this is likely to be a causative relationship. Reduced TNX expression could disturb deposition of collagen (Mao et al. 2002) and the elastic fiber network (Burch et al. 1997), as has been shown for complete TNX deficiency, resulting in increased laxity of ligaments and tendons. TNXB haploinsufficiency is dominantly inherited and appears to produce clinical findings primarily in women, consistent with clinical descriptions of HT-EDS. Although we identified inactivating TNX mutations in only 2.5% of this cohort with HT-EDS, 7.5% had serum TNX levels low enough to affect collagen metabolism. The present study demonstrates that TNXB haploinsufficiency is associated with HT-EDS and suggests that locus heterogeneity exists for this disorder, as it does for other types of EDS (Byers 1994).

Prognostic factors in the treatment of alopecia areata with diphenylcyclopropenone

One hundred thirty-nine patients with alopecia areata were treated with diphenylcyclopropenone. Before treatment, 85 patients had subtotal or total hair loss (greater than 90% bald area) and in the remaining patients scalp involvement was between 40% and 90%. The following three factors were found to be of prognostic significance: type of alopecia areata as documented before treatment, duration of the disease before therapy, and presence of nail changes. Other factors such as age at onset, sex, presence of atopic features, the extent of variation in the range of diphenylcyclopropenone concentrations during treatment, and sleep disturbances caused by pruritus did not influence the prognosis significantly.

The Tetraspanin CD37 Orchestrates the alpha4beta1 Integrin-Akt Signaling Axis and Supports Long-Lived Plasma Cell Survival

Antibody-producing B cells require CD37-dependent integrin signaling for long-term survival. CD37 Stimulates Plasma Cell Survival To generate immunological memory, B cells with high-affinity immunoglobulin receptors proliferate and differentiate in germinal centers in the spleen to produce memory B cells and long-lived antibody-secreting cells known as plasma cells. van Spriel et al. found that mice deficient in the tetraspanin protein CD37 had defective antibody production and decreased numbers of germinal center B cells compared to those in wild-type mice, which was a result of enhanced apoptosis. Survival signals in B cells were initiated by engagement of the integrin α4β1 and activation of the downstream kinase Akt. In the absence of CD37, integrin clustering and function were impaired, and activation of the Akt survival pathway was defective. Thus, long-lived plasma cells rely on the tetraspanin CD37 to enable integrin-Akt survival signaling. Signaling by the serine and threonine kinase Akt (also known as protein kinase B), a pathway that is common to all eukaryotic cells, is central to cell survival, proliferation, and gene induction. We sought to elucidate the mechanisms underlying regulation of the kinase activity of Akt in the immune system. We found that the four-transmembrane protein CD37 was essential for B cell survival and long-lived protective immunity. CD37-deficient (Cd37−/−) mice had reduced numbers of immunoglobulin G (IgG)–secreting plasma cells in lymphoid organs compared to those in wild-type mice, which we attributed to increased apoptosis of plasma cells in the germinal centers of the spleen, areas in which B cells proliferate and are selected. CD37 was required for the survival of IgG-secreting plasma cells in response to binding of vascular cell adhesion molecule 1 to the α4β1 integrin. Impaired α4β1 integrin–dependent Akt signaling in Cd37−/− IgG-secreting plasma cells was the underlying cause responsible for impaired cell survival. CD37 was required for the mobility and clustering of α4β1 integrins in the plasma membrane, thus regulating the membrane distribution of α4β1 integrin necessary for activation of the Akt survival pathway in the immune system.

Cellular and plasma adriamycin concentrations in long-term infusion therapy of leukemia patients

SummaryTo determine whether long-term adriamycin (ADM) infusions resulted in cellular ADM concentrations at least comparable to those observed after bolus injections, ADM cellular and plasma concentrations were measured in 18 patients with leukemia. ADM was administered at 30 mg/m2 per day for 3 days, either as bolus injections or as 4-, 8-, or 72-h infusions. Negligible accumulation of plasma ADM was observed. Peak plasma ADM concentrations after bolus injections were 1640±470 ng/ml (n=7). Maximum levels were 176±34 ng/ml during 4-h infusion (n=5); 85±50 ng/ml during 8-h infusion (n=4); and 47±5 ng/ml (n=2) after 72-h infusion. ADM concentrations in nucleated blood and bone marrow cells correlated well (r=0.82, n=47). ADM accumulated in leukemic cells up to 30–100 times the plasma concentrations. The shorter the administration time-span, the higher the peak leukemic cell concentration and the greater the loss of drug immediately after the end of the administration. The final cellular ADM half-life was approximately 85–110 h. After long-term infusion and bolus injection of the same dose, similar areas under the curve for plasma or leukemic blast cell ADM concentrations were attained. Since comparable therapeutic efficacy was observed in all regimens, the antileukemic effect appeared not to be related to the peak plasma concentrations, while acute toxicity phenomena decreased with increasing duration of the infusion. Long-term ADM infusion deserves more attention in the treatment of patients with anthracyclines.

Frequency of moles as a key to melanoma incidence

Subjects with light skin complexion are especially prone to develop melanoma. Furthermore, individuals with large numbers of moles are also at increased risk of developing melanoma. We studied the association between these two markers of melanoma in a group of healthy white people: 116 children 6 to 9 years of age, 78 children 10 to 13 years of age, and 133 medical and nursing students 18 to 30 years of age. Moles were counted on the chest, back, and lower legs. Skin complexion was established with the use of a scoring system based on burning/tanning ability, eye and hair color, and freckling tendency. A multifactorial statistical analysis was performed. Average mole counts increased with age (p less than 0.0001). Male subjects had more nevi on the trunk than female subjects, whereas counts on the lower legs were higher in female than in male subjects (p = 0.0001). Skin complexion was an important denominator of mole proneness; subjects with a light complexion had higher mean counts than those with a dark complexion (p = 0.0001). The present study shows that a significant association exists between skin complexion phenotype and numbers of moles. The pattern of the appearance of pigmented nevi roughly correlates with the sex and site distribution of melanoma.

Flow cytometric analysis of the recruitment of G0 cells in human epidermis in vivo following tape stripping

Abstract. Tape stripping of human skin elicits a proliferative response of a synchronously‐dividing group of cells. The progress of this cohort of cells has been monitored using two windows in the cell cycle, one located in mid‐S phase and the other centred around G2+ M. The cellular DNA is measured with flow cytometry, the windows are defined by two ranges in the DNA histogram.

Leukemic cell and plasma daunomycin concentrations after bolus injection and 72 h infusion

SummaryThe effect of the duration of daunomycin (DNM) infusion on leukemic cell drug concentrations was evaluated. Cellular and plasma DNM concentrations were measured in 20 patients with acute non-lymphocytic leukemia. DNM 45 mg/m2 was administered either as a bolus injection or as a 4-, 8- or 72-h constant-rate infusion during 3 consecutive days. Peak plasma DNM levels amounted to 227±116 ng/ml after bolus injection and were only 16±6 ng/ml after 72-h DNM infusions. Terminal plasma DNM half-lives were 14±4 h. Peak leukemic cell DNM concentrations at the 3rd day of administration were 16810±2580 ng/109 cells (bolus injections) and 10310±5510 ng/109 cells (72-h infusions). The areas under the cellular curve were similar and independent of the duration of the DNM infusion. Peak leukemic cell daunomycinol (DNMol) concentrations were respectively 3500 ± 1600 ng/109 cells and 2850±1720 ng/109 cells. Cellular DNM terminal half-life was 13±4 h. DNM concentrations in nucleated blood and bone marrow cells correlated well (r=0.93, n=26). Long-term infusion produced less severe side effects. Therapeutic efficacy was maintained during long-term infusion.

Differences between ulcerated and non-ulcerated hemangiomas, a retrospective study of 465 cases.

Our purpose was to get better insight into the ulceration of hemangiomas, by comparing patient characteristics of non-ulcerated hemangiomas with hemangiomas with active or past ulceration. A retrospective analysis was performed of files of patients who visited the Radboud University Medical Centre Nijmegen (UMCN), the Netherlands, between 1997 and 2007 for one or more infantile hemangiomas. The medical records of 465 patients were reviewed. Twenty three percent of the patients were diagnosed with ulceration. The size of ulcerated hemangiomas was significantly larger (28.6 cm2 vs. 6.0 cm2, p < 0.05). Predilection areas for ulceration were the head-neck region and the anogenital region. Ulceration was significantly most frequently seen in hemangiomas with a superficial (epidermal) component (98.5%, p < 0.05) and a segmental distribution (29.3%, p < 0.05). Ulceration most frequently took place during the proliferation phase of the hemangioma (83.1%). In the whole study population the male to female ratio was 1:2 compared to a tendency to more girls (1:3) for the group with ulcerated hemangiomas (p = 0.08). We conclude that larger, more superficial hemangiomas in areas more susceptible to trauma and contamination were more likely to ulcerate. This study contributes to the possibility of assessing the likelihood of ulceration in an individual patient.

A low but functionally significant MDR1 expression protects primitive haemopoietic progenitor cells from anthracycline toxicity

Pgp is expressed on normal haemopoietic progenitor cells. The significance of the efflux pump in protecting normal progenitors for anthracycline toxicity is not defined and is the subject of this study. Pgp was measured in CD34+ progenitors with a rhodamine efflux assay. A high efflux, modulated by verapamil, was only found in a distinct subpopulation (20–30%). Pgp measured by the monoclonal antibody antibody (MoAb) MRK‐16 was low in the rhodamine dull, but significantly (P < 0.04) higher than in the rhodamine bright cells. Reverse transcriptase polymerase chain reaction (RT‐PCR) of MDR1 mRNA showed a very weak signal in both populations. In a single‐cell clonogenic assay, rhodamine dull cells appeared less sensitive to anthracyclines (IC50 daunorubicin 0.005 μg/ml; adriamycin 0.03 μg/ml) compared to rhodamine bright cells (IC50 daunorubicin 0.0025 μg/ml; adriamycin 0.01 μg/ml). Furthermore, verapamil significantly (P < 0.05) potentiated anthracycline toxicity only in the rhodamine dull cells, proving its Pgp‐specific modulating effect. Rhodamine dull cells gave larger and more mixed colonies compatible with a more primitive origin. Although detection with MoAbs and RT‐PCR revealed a low Pgp level, functionally this Pgp appeared to be very important in protecting primitive progenitors against anthracycline toxicity. This protection can be jeopardized by administration of Pgp modulators.