Mitotically active proliferative nodule arising in a giant congenital melanocytic nevus: a diagnostic pitfall.

Proliferative (cellular) nodules (PN) which mimic malignant melanoma clinically and histologically are described in congenital melanocytic nevi (CMN) and may pose significant diagnostic challenges. We report the case of a 10-day-old male with a giant congenital nevus involving the neck, upper chest, back, and left shoulder containing several nodular lesions, some crusted. Biopsy of a nodule revealed densely packed nevus cells with hyperchromatic round to oval and occasionally irregularly shaped nuclei. There was no necrosis or pushing border, and the nodule blended with the adjacent nevus; however, the lesion demonstrated a significant number of mitoses (27 per mm2) and a 60% labeling index with Ki-67. Further analysis by fluorescence in situ hybridization (FISH) with a 4-color probe set targeting 6p25, 6q23, 11q13, and centromere 6 revealed increased chromosomal copy numbers of all 4 probes, which was interpreted as evidence of polyploidy. In addition, analysis of DNA copy number changes using a single nucleotide polymorphism microarray (Affymetrix, Santa Clara, CA) showed no chromosomal aberrations. The diagnosis of PN in a giant congenital nevus was eventually rendered. At 13-month follow-up, the nodules showed no evidence of growth. Our case illustrates that PNs in the neonatal period might demonstrate extreme mitotic activity. This feature is worrisome when encountered in melanocytic lesions; however, it should not trigger by itself a diagnosis of melanoma in the absence of other histologic criteria of malignancy. In addition, we document polyploidy by FISH in PN, which can potentially be misinterpreted as a FISH-positive result.

Absence of time-dependent artesunate pharmacokinetics in healthy subjects during 5-day oral administration.

AIM: The aim of the present study was to investigate the existence of time-dependent pharmacokinetics of artesunate (ARS) during 5 consecutive days of oral administration to 10 healthy Vietnamese subjects (aged 21-52 years and weighing 49-90 kg). METHODS: Each volunteer received 200 mg oral doses of ARS once daily for 5 consecutive days. Blood samples (3 ml each) were collected on days 1 and 5 at 0 (before dosing), 0.5, 1, 2, 3, 4, and 6 h after drug administration. During days 2, 3, and 4, the same volumes of blood were collected at 0, 1, 2, and 4 h after dosing. Plasma ARS and dihydroartemisinin (DHA) were measured using high-performance liquid chromatography-mass spectrometry (LC-MS). RESULTS: Results did not show evidence of time-dependency for ARS or the active plasma metabolite DHA. There were no differences in the concentrations of ARS and DHA at all sampling times on days 1 and 5. In addition, the pharmacokinetics of both compounds were similar on days 1 and 5. This finding confirms that the enzyme auto-induction in drug metabolism may not be characteristic of the endoperoxide sesquiterpene antimalarial group.

Evaluation of biological and economic efficiency of smallholder pig production systems in North Vietnam.

This study evaluates smallholder pig production systems in North Vietnam, comparing a semi-intensive system near a town with good market access, where a Vietnamese improved breed has replaced the indigenous pig breed, and an extensive system away from town, where the indigenous breed still prevails. Fieldwork was conducted in 64 households in four villages. Repeated farm visits yielded 234 structured interviews. Data were analysed by linear models and non-parametric tests. Production inputs and outputs were quantified, and feed use efficiency and economic efficiency were assessed. The gross margin was higher for semi-intensive production with the improved breed, while the benefit-cost ratio was higher under extensive conditions with the indigenous breed. The net benefit did not differ between systems. Twenty-four per cent of farmers yielded a negative net benefit. In one village under extensive conditions, live weight output from indigenous sows with crossbred offspring compared positively with the output from semi-intensive production with improved genotypes, but was associated with high inputs, making production inefficient. Results indicate that improved genotypes might not be an efficient production alternative for saving-oriented production with limited resource supply. Suitability of evaluation parameters, farmers' production aims, and factors impacting the production success in different systems are discussed.

Identification and quantification of subsidies relevant to the production of local and imported pig breeds in Vietnam.

Livestock diversity contributes in many ways to human survival and well-being, while its loss reduces options for attaining sustainable agriculture and universal food security. The current rapid rate of loss of this diversity is the result of a number of underlying factors. While in some cases changes in production systems and consumer preferences reflect the natural evolution of developing economies and markets, in other cases production systems, breed choice and consumer preferences have been distorted by local, national and international policy. In the context of a widespread threat to local pig breeds in Vietnam, this paper identifies and quantifies the level of agricultural subsidies that are currently contributing to this process of breed substitution. Producer subsidies-which tend to improve the competitiveness of imported breeds and their crosses over local breeds--are shown to be considerable, and mitigating measures are now urgently needed to avoid an irreversible loss of livestock diversity.

Microsatellite analysis of genetic diversity of the Vietnamese sika deer (Cervus nippon pseudaxis).

The Vietnamese sika deer (Cervus nippon pseudaxis) is an endangered subspecies of economic and traditional value in Vietnam. Most living individuals are held in traditional farms in central Vietnam, others being found in zoos around the world. Here we study the neutral genetic diversity and population structure of this subspecies using nine microsatellite loci in order to evaluate the consequences of the limited number of individuals from which this population was initiated and of the breeding practices (i.e., possible inbreeding). Two hundred individuals were sampled from several villages. Our data show both evidence for limited local inbreeding and isolation by distance with a mean F(ST) value of 0.02 between villages. This suggests that exchange of animals occurs at a local scale, at a rate such that highly inbred mating is avoided. However, the genetic diversity, with an expected heterozygosity (H(e)) of 0.60 and mean number of alleles (k) of 5.7, was not significantly larger than that estimated from zoo populations of much smaller census size (17 animals sampled; H(e) = 0.65, k = 4.11). Our results also suggest that the Vietnamese population might have experienced a slight bottleneck. However, this population is sufficiently variable to constitute a source of individuals for reintroduction in the wild in Vietnam.

Indomethacin stimulates glucose production in adults with uncomplicated falciparum malaria.

In healthy subjects, basal hepatic glucose production is (partly) regulated by paracrine intrahepatic factors. It is unknown if these paracrine factors also influence basal glucose production in infectious diseases with increased glucose production. We compared the effects of 150 mg indomethacin (n = 9), a nonendocrine stimulator of glucose production in healthy adults, and placebo (n = 7) on hepatic glucose production in Vietnamese adults with uncomplicated falciparum malaria. Glucose production was measured by primed, continuous infusion of [6,6-2H2]glucose. After indomethacin, the plasma glucose concentration and glucose production increased in all subjects from 5.3 +/- 0.1 mmol/L to a maximum of 7.1 +/- 0.3 mmol/L (P < .05) and from 17.6 +/- 0.8 micromol x kg(-1) x min(-1) to a maximum of 26.2 +/- 2.5 micromol x kg(-1) x min(-1) (P < .05), respectively. In the control group, the plasma glucose concentration and glucose production declined gradually during 4 hours from 5.4 +/- 0.2 mmol/L to 5.1 +/- 0.1 mmol/L (P < .05) and from 17.1 +/- 0.8 micromol x kg(-1) x min(-1) to 15.1 +/- 1.0 micromol x kg(-1) x min(-1) (P < .05), respectively. There were no differences in plasma concentrations of insulin, counterregulatory hormones, or cytokines between the groups. We conclude that indomethacin administration results in a transient increase in glucose production in patients with uncomplicated falciparum malaria in the absence of changes in plasma concentrations of glucoregulatory hormones or cytokines. Thus, this study indicates that in uncomplicated falciparum malaria, the rate of basal hepatic glucose production is also regulated by paracrine intrahepatic factors.

Glucose production and gluconeogenesis in adults with uncomplicated falciparum malaria.

Although glucose production is increased in severe malaria, the influence of uncomplicated malaria on glucose production is unknown. Therefore, we measured in eight adult Vietnamese patients with uncomplicated falciparum malaria and eight healthy Vietnamese controls glucose production (by infusion of [6,6-2H2]glucose) and the fractional contribution of gluconeogenesis (by oral ingestion of 2H2O); glycogenolysis was calculated as the difference between the two. After 20 h of fasting, plasma glucose was 4.7 +/- 0.2 mmol/l in the patients and 4.3 +/- 0.2 mmol/l in the controls (not significant). Glucose production was approximately 25% higher in the patients (16.9 +/- 1.3 vs. 13.4 +/- 0.3 mumol.kg-1.min-1, P = 0.01). Fractional and absolute gluconeogenesis were increased in the patients (approximately 87 vs. approximately 59%, P < 0.001; and 14.6 +/- 1.3 vs. 7.9 +/- 0.2 mumol.kg-1.min-1, P < 0.001, respectively). The contribution of glycogenolysis to total glucose production was decreased in the patients: 2.3 +/- 0.5 vs. 5.5 +/- 0.4 mumol.kg-1.min-1 (P < 0.002). In conclusion, in adult patients with uncomplicated falciparum malaria, glucose production is increased by approximately 25% due to an increased rate of gluconeogenesis, whereas glycogenolysis is decreased. The mechanism by which these changes occur is uncertain. However, counterregulatory hormone and cytokine concentrations were increased in the patients.

Blood levels of DDT and breast cancer risk among women living in the north of Vietnam.

A positive association has been reported between elevated tissue organochlorines (p,p'-DDT/p,p'-DDE, PCBs, dioxins) and breast cancer in some case-control studies and occupational cohort studies. We previously reported high serum levels of p,p'-DDT and its metabolite p,p'-DDE in women living throughout Vietnam. We report here the results of a small hospital-based case-control study examining the association between blood levels of p,p'-DDT/p,p'-DDE and the risk of invasive breast cancer among residents of the north of Vietnam-an area where insecticides such as p,p'-DDT have been heavily used in the recent past. The study was conducted among patients admitted to a single hospital in the capital city of Hanoi in 1994. Study subjects were 21 women newly diagnosed with invasive adenocarcinoma of the breast, who served as cases, and 21 women of similar age with fibrocystic breast disease, who served as controls. No increase was evident in the relative risk of breast cancer with increasing tertiles of serum concentration of the compounds of interest, even after adjustment for major potential confounders, such as age at menarche, parity, history of lactation, and body weight. These results suggest that recent and past exposure to p,p'-DDT does not play an important role in the etiology of breast cancer among women living in a country with a tropical climate where insecticide use for mosquito control is common.

Agent Orange and the Vietnamese: the persistence of elevated dioxin levels in human tissues.

OBJECTIVES: The largest known dioxin contamination occurred between 1962 and 1970, when 12 million gallons of Agent Orange, a defoliant mixture contaminated with a form of the most toxic dioxin, were sprayed over southern and central Vietnam. Studies were performed to determine if elevated dioxin levels persist in Vietnamese living in the south of Vietnam. METHODS: With gas chromatography and mass spectroscopy, human milk, adipose tissue, and blood from Vietnamese living in sprayed and unsprayed areas were analyzed, some individually and some pooled, for dioxins and the closely related dibenzofurans. RESULTS: One hundred sixty dioxin analyses of tissue from 3243 persons were performed. Elevated 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) levels as high as 1832 ppt were found in milk lipid collected from southern Vietnam in 1970, and levels up to 103 ppt were found in adipose tissue in the 1980s. Pooled blood collected from southern Vietnam in 1991/92 also showed elevated TCDD up to 33 ppt, whereas tissue from northern Vietnam (where Agent Orange was not used) revealed TCDD levels at or below 2.9 ppt. CONCLUSIONS: Although most Agent Orange studies have focused on American veterans, many Vietnamese had greater exposure. Because health consequences of dioxin contamination are more likely to be found in Vietnamese living in Vietnam than in any other populations, Vietnam provides a unique setting for dioxin studies.

Modulation of the expression of CD5 antigen on the surface of human peripheral B lymphocytes.

In this study, we investigated the expression of CD5 molecules on the surface of mature human peripheral B lymphocytes. Human CD5+ B cells were isolated from tonsils and peripheral blood. Their terminal differentiation into plasma cells was induced with IL-2. In the presence of this lymphokine, a subset of CD5+ B cells ceased to express CD5 proteins on their surface. When CD5+ B cells and non-B cells were removed from the suspension. CD5 antigens were spontaneously reexpressed on the surface of CD5- B cells. This suggests that the CD5- phenotype of a subset of B cells in these suspensions resulted from pressure(s) exerted on them by the other cellular components of the suspensions. B cells which have reexpressed membrane CD5 in the absence of CD5+ B cells and non-B cells retained their ability to reprogress to CD5- phenotype and to undergo terminal differentiation into plasma cells when stimulated with IL-2. A short exposure (4 hr) of CD5- B cells to 5 nM IL-2 resulted in the loss of their capability to spontaneously reexpress surface CD5 in the absence of other lymphoid cells. Taken together, these data suggest that the expression of CD5 antigens on B cell membrane is governed by a network-type balance between all cellular compartments within the suspension. Fluctuations of this equilibrium results in the shuttling of B cells between CD5+ and CD5+ phenotypes until they become irreversibly engaged in the terminal differentiation pathway. The interconversion CD5+<==>CD5- on B cell membrane does not argue for CD5 molecule as the marker of a distinct B cell lineage.

Correlation between dioxin levels in adipose tissue and estimated exposure to Agent Orange in south Vietnamese residents.

To permit new epidemiologic studies of the effects of dioxin on humans in Vietnam, we evaluated a model for quantifying exposure to Agent Orange (exposure index) based on the residential histories of 27 Vietnamese subjects and on information about spraying from the U.S. Army records (Herbs Tape) and compared this index to the dioxin levels measured in the subjects' adipose tissue. The mean dioxin level was 7.8 ppt, and dioxin and furan isomer profiles were similar to those already reported in industrialized countries. In addition, there was a highly significant correlation between the levels of almost all the isomers, whatever their degree of chlorination. For the group of 27 subjects, we found a Pearson correlation coefficient of 0.36 (P = 0.07) between the dioxin levels and the exposure index after log-transformation of both variables. When the analysis was restricted to the 22 subjects with a positive exposure index, the Pearson correlation coefficient rose to 0.50 (P = 0.02). We conclude that despite the limitations and power conditions of the study, this result is encouraging because it will be useful for future epidemiologic studies in Vietnam.

High-affinity receptors for interleukin-2 are not required for the induction of human unstimulated B lymphocyte differentiation by this lymphokine.

We have reported that one of the currently known receptors for interleukin-2 (IL-2), the p70 protein, is constitutively expressed on resting T lymphocyte membrane. We demonstrated that exposure of these cells to high concentrations of IL-2 resulted in the transcription of genes whose expression occurred early during cell activation such as cmyc, cmyb protooncogenes, and the Tac gene itself. IL-2 is thought to exert its biological effects by binding to its high-affinity receptors on cell membrane. Recent studies using B cell lines emphasized that within the high-affinity receptor complexes, p55 serves to allow high-affinity binding, and p70, to transduce signals. In this study, we prepared human unstimulated B cells devoid of detectable Tac antigen, and consequently, of the high-affinity receptor complexes. We designed experiments such that no in vitro de novo expressed receptors, if any, could interact with IL-2, so that any biologic events triggered by IL-2 must have been mediated in the absence of the high-affinity receptors. Under these conditions, we demonstrated that a short and unique exposure (1 hr) of these cells to high concentrations (5 nM) of IL-2 allowed its binding to cell membrane and resulted in a competent signal leading to the generation of a majority of 25S Tac mRNA, and a progression signal allowing B cell terminal differentiation into plasma cells.

An enhancer associated with the mouse immunoglobulin lambda 1 gene is specific for lambda light chain producing cells.

We have developed a system to study transcriptional regulation of the lambda immunoglobulin gene in a natural setting -- lambda light chain producing lymphoid cells. This assay system has allowed the detection of an enhancer element located 3' of the lambda gene coding sequence. The enhancer can stimulate transcription from the lambda promoter as well as from other immunoglobulin and unrelated promoters. Like all enhancers, the lambda enhancer can function in either orientation with respect to a promoter, but it is significantly more active in one orientation than in the other. The lambda enhancer is unusual in spanning at least 4000 bp of DNA sequence and containing several distinct subelements that have independent enhancer activity. The enhancer is also remarkable because it functions in lambda light chain producing cells but not in kappa chain producing cells. This fact can be interpreted to support a model of immunoglobulin gene rearrangement in which rearrangement follows and depends on transcriptional activation.

Transfection of an immunoglobulin kappa gene into mature human B lymphocytes.

We show in this report that the transcription induced by interleukin-2 or pokeweed mitogens of the kappa MOPC 41 immunoglobulin light-chain gene transfected into primary human or murine B lymphocytes initiates from a previously unobserved start site about 26 base pairs upstream of the start site used in myeloma cell lines.

Direct activation of human resting T cells by IL 2: the role of an IL 2 receptor distinct from the Tac protein.

High concentrations of interleukin 2 (IL 2) were shown to produce a delayed but pronounced proliferation of purified resting T cells in the apparent absence of other activation signals. Because these stimulatory effects of IL 2 occurred in the absence of detectable Tac+ cells, the possibility that IL 2 might be initially interacting with an IL 2 binding protein distinct from the Tac protein was studied. Chemical cross-linking studies with 125I-IL 2 revealed the presence of an IL 2 binding protein distinct from the Tac protein on the surface of these unstimulated T cells. This second IL 2 receptor has an estimated molecular size of 70,000 daltons, lacks reactivity with the anti-Tac antibody, and appears to be identical to the p70 protein recently proposed as a component of the high affinity IL 2 receptor. Scatchard analysis of IL 2 binding assays performed with the unactivated T cells revealed approximately 600 to 700 p70 sites per cell and an apparent Kd of 340 pM. These data indicate that the p70 protein present on resting T cells binds IL 2 with an intermediate affinity compared with the previously recognized high and low affinity forms of the receptor and may account for the high concentration of IL 2 needed to induce resting T cell proliferation. To investigate the early biologic consequences of IL 2 binding to the p70 protein, potential changes in the expression of genes involved in T cell activation were examined. Northern blotting revealed the rapid induction of c-myc, c-myb, and Tac mRNA after stimulation of resting T cells with a high concentration of IL 2. The anti-Tac antibody did not inhibit IL 2 induced expression of these genes, suggesting that the p70 protein rather than the Tac antigen or the high affinity IL 2 receptor complex mediated this signal. However, in contrast to these early activation events, the anti-Tac antibody significantly inhibited IL 2 induced T cell proliferation. This finding implicates the high affinity form of the IL 2 receptor in the proliferative response of the IL 2 activated T cells. Thus these data support a two step model for the induction of resting T cell proliferation by high doses of IL 2 involving the initial generation of an activation or "competence" signal through the p70 protein and a subsequent proliferation or "progression" signal through the high affinity form of the receptor.

Synergism between immunoglobulin enhancers and promoters.

Enhancers are DNA sequences that stimulate transcription from eukaryotic promoters. This stimulatory effect can be exerted over large distances and from a position either 5' or 3' of a promoter. Enhancers have been found in the genomes of many viruses, and in some cellular genes such as those encoding immunoglobulin heavy chain and kappa light chain. An important feature of both viral and cellular enhancers is the ability of each enhancer to stimulate transcription from many promoters other than the one with which it is found associated. However, the question of whether cellular enhancers stimulate their 'own' promoter more efficiently than other promoters has apparently not been investigated. We show here that the kappa light-chain enhancer stimulates a kappa promoter about 20-fold more than it stimulates either the simian virus 40 (SV40) early promoter or a metallothionein (MT) promoter, two promoters that are very sensitive to other enhancers. Similarly, the heavy-chain enhancer stimulates a heavy-chain promoter much more than it stimulates the SV40 and MT promoters. This synergism between immunoglobulin enhancers and promoters might be due to the action of a protein that binds specifically to each of the regulatory elements.

Recombinant interleukin-2-induced polyclonal proliferation of in vitro unstimulated human peripheral blood lymphocytes.

Human peripheral blood mononuclear cells as well as T-cell-enriched or T-cell-depleted populations were found to proliferate in response to recombinant interleukin-2 (IL-2) in vitro in the absence of a lectin preactivation signal. This proliferative response was detected at Day 2, peaked at Day 5, and was dependent on the concentration of IL-2 used. At the initiation of culture, these cells did not appear to be activated as determined by the expression of Tac antigens. In cultures of unfractionated T-cell-enriched suspensions, high concentrations of IL-2 resulted in preferential expansion of the OKT8+ population, although both OKT4+ and OKT8+ cells proliferated in response to IL-2 when cultured alone. These studies demonstrate that human lymphocytes obtained by standard fractionation procedures from peripheral blood are capable of proliferation in response to IL-2 without in vitro preactivation signals given by the addition of mitogens or antigens to cultures. These findings suggest that in vivo IL-2, in the absence of other exogenous stimuli, may directly influence immune responses and thus may have a potential role as a clinical immunopharmacologic agent.

Distribution of Ig classes and IgG subclasses among human B cells activated by Nocardia opaca-delipidated cell mitogen or by pokeweed mitogen.

The relative proportions of cells synthesizing the three major Ig classes or one of the four IgG subclasses in cultures stimulated with pokeweed mitogen (PWM) or Nocardia-delipidated cell mitogen (NDCM) were investigated. In cultures of human peripheral blood mononuclear cells (PB MNC) stimulated with PWM, the number of IgG-containing cells (CC) was higher than the number of IgM-CC, and a substantial number of IgA-CC was found. Conversely, in NDCM-stimulated PB MNC cultures IgM-CC outnumbered IgG-CC and only few IgA-CC were detected. In those cultures, the removal of T cells resulted in an increase in the number of IgM-CC concomitant with a decrease in the number of IgG-CC. A substantial number of cells containing simultaneously IgG or IgA in addition to IgM could be found in PWM-stimulated cultures. These cells were virtually absent in NDCM-stimulated cultures. The relative proportions of IgG subclass-CC were IgG1-CC greater than IgG2-CC greater than IgG3-CC greater than or equal to IgG4-CC in PWM-stimulated and IgG2-CC greater than IgG1-CC greater than IgG3-CC greater than or equal to IgG4-CC in NDCM-stimulated cultures. The removal of T cells from NDCM-stimulated cultures did not result in major alteration of this distribution. The role of T cells and of the genomic order of the Igh-C genes in their phenotypic expression triggered in vitro by PBA is discussed.

Modulation of polyclonally activated human peripheral B cells by aggregated IgG and by IgG-binding factors: differential effect on IgG subclass synthesis.

The regulation of IgG subclass production by polyclonally activated human B cells was investigated by using two systems previously shown to selectively suppress the generation of IgG-containing cells (CC) but not that of IgMCC or IgACC. The first one involved a brief exposure of peripheral blood mononuclear cells (PBMNC) to heat-aggregated human IgG (Agg-IgG) followed by repeated washings and culture with untreated autologous PBMNC. The second one was achieved by addition of human IgG-binding factor(s) (IgGBF) prepared by affinity chromatography from supernatants of unstimulated PBMNC. Pokeweed mitogen (PWM) and Nocardia opaca delipidated cell mitogen (NDCM) were used as polyclonal B cell activators. The latter can induce the terminal differentiation of peripheral B lymphocytes into plasma cells in the absence of helper T cells. After 6 days of culture, the number of cells containing IgM, IgG, or IgA was determined by direct immunofluorescence, and that of cells containing IgG1, IgG2, IgG3, or IgG4 was determined by indirect immunofluorescence with the use of subclass-specific monoclonal antibodies. After stimulation with PWM, exposure of PBMNC to Agg-IgG resulted in a selective diminution of the number of IgG4CC. With NDCM-stimulated cultures the same procedure induced a selective suppression of the generation of IgG2CC and IgG4CC. Conversely, the addition of IgGBF at the third day of culture was found to induce a 30 to 40% decrease in the number of cells containing each of the four IgG subclasses. Because of their differential pattern of IgG subclass suppression, Agg-IgG and IgGBF are likely to trigger distinct regulatory pathways.