Frequent ongoing T-cell receptor rearrangements in childhood B- precursor acute lymphoblastic leukemia: implications for monitoring minimal residual disease

Crosslineage T-cell receptor delta (TCR delta) rearrangements are widely used as tumor markers for the follow up of minimal residual disease in childhood B-precursor acute lymphoblastic leukemia (ALL) by polymerase chain reaction (PCR). The major drawback of this approach is the risk of false-negative results due to clonal evolution. We investigated the stability of V delta 2D delta 3 rearrangements in a group of 56 childhood B-precursor ALL patients by PCR and Southern blot analysis. At the PCR level, V delta 2D delta 3-to-J alpha rearranged subclones (one pathway for secondary TCR delta recombination) were demonstrated in 85.2% of V delta 2D delta 3-positive patients tested, which showed that small subclones are present in the large majority of patients despite apparently monoclonal TCR delta Southern blot patterns. Sequence analysis of V delta 2D delta 3J alpha rearrangements showed a biased J alpha gene usage, with HAPO5 and J alpha F in 26 of 32 and 6 of 32 clones, respectively. Comparison of V delta 2D delta 3 rearrangement status between diagnosis and first relapse showed differences in seven of eight patients studied. In contrast, from first relapse onward, no clonal changes were observed in six patients studied. To investigate the occurrence of crosslineage TCR delta rearrangements in normal B and T cells, fluorescence-activated cell sorter-sorted peripheral blood CD19+/CD3- and CD19-/CD3+ cell populations from three healthy donors were analyzed. V delta 2D delta 3 rearrangements were detected at low frequencies in both B and T cells, which suggests that V delta 2-to-D delta 3 joining also occurs during normal B-cell differentiation. A model for crosslineage TCR delta rearrangements in B-precursor ALL is deduced that explains the observed clonal changes between diagnosis and relapse and is compatible with multistep leukemogenesis of B-precursor ALL.     

Vascular dissemination of tumor cells in relation to temperature-dependent metastasis in frogs

Metastasis is a temperature-related phenomenon in the North American leopard frog (Rana pipiens) affected with the Lucké renal adenocarcinoma. The frog is a poikilothermic vertebrate whose internal body temperature closely follows that of the environment, and tumor-bearing frogs living in a warm (28 degrees C) environment have a much higher incidence of metastasis (greater than 75%) than those in cold (4 degrees C) conditions (less than 6%). In this investigation it was found that Lucké tumor cells labeled with fluorescein isothiocyanate could be detected in frozen sections of all organs examined within 15 minutes, regardless of whether the hosts were adapted to a warm or a cold environment. Separate experiments involving inoculation of isotope-labeled tumor cells demonstrated that large numbers of cells arrived in the liver and other organs of animals kept at either end of the temperature range. These findings show that the infrequency of metastasis by Lucké adenocarcinomas in chilled frogs is not due to failure of dissemination of tumor cells consequent upon temperature-mediated changes in blood viscosity or flow patterns. Thus they establish the important baseline that the effects of temperature on metastasis in this vertebrate are exerted directly on the tumor cells and/or the internal environment of the host and that the system therefore provides new opportunities for probing the biology and biochemistry of tumor metastasis.     

A rapid digestion method for analysis of nickel compounds in tissue by electrothermal atomic absorption spectrometry

Quantification of nickel in animal soft tissue is of toxicological interest. A digestion method applying the use of microwave ovens for irradiating samples in Teflon digesters was developed. An acid mixture containing nitric acid (16 M, 1.0 ml g-1 tissue), hydrochloric acid (6 M, 0.5 ml g-1 tissue) and H2O2 (30%, 1.0 ml g-1 tissue) and irradiation at 600 W for 5 min were required for complete dissolution of tissue matrices and nickel compounds. Analyses of Ni in National Bureau of Standards Reference Material 1566 oyster tissue gave 0.87 +/- 0.24 micrograms g 1(mean +/- SD, n = 5), which was in agreement with the NBS certified value of 1.03 +/- 0.19 micrograms g-1. Recoveries of 1-300 micrograms Ni added as nickel sulfate (highly soluble), nickel subsulfide (moderately soluble in biological fluids and acid) or nickel oxide (green high-temperature oxide, low solubility in biological fluids and acid) to lung, liver, lymph node and kidney were quantitative, except in the case of nickel sulfate added to kidney, where recovery was less than quantitative for 1-10 micrograms Ni. The method appears effective for digestion of a variety of tissues requiring Ni analyses.     

Cost comparison of unit dose and traditional drug distribution in a long-term-care facility

Unit dose and traditional drug distribution systems were compared in a 352-bed long-term-care facility by analyzing nursing time, medication-error rate, medication costs, and waste. Time spent by nurses in preparing, administering, charting, and other tasks associated with medications was measured with a stop-watch on four different nursing units during six-week periods before and after the nursing home began using unit dose drug distribution. Medication-error rate before and after implementation of the unit dose system was determined by patient profile audits and medication inventories. Medication costs consisted of patient billing costs (acquisition cost plus fee) and cost of medications destroyed. The unit dose system required a projected 1507.2 hours less nursing time per year. Mean medication-error rates were 8.53% and 0.97% for the traditional and unit dose systems, respectively. Potential annual savings because of decreased medication waste with the unit dose system were $2238.72. The net increase in cost for the unit dose system was estimated at $615.05 per year, or approximately $1.75 per patient. The unit dose system appears safer and more time-efficient than the traditional system, although its costs are higher.     

Host response toBothrops asper snake venom

As part of the characterization of the host reactivity to the venom ofBothrops asper, we investigated the inflammatory responses in the mouse footpad model. The subcutaneously injected venom induced a rapid increase of serum IL-6 concentration, which peaked between 3 and 6 h and returned to normal values at 12 h. In contrast, serum TNF- and IL-1 were not detectable at any time point studied. A myotoxic phospholipase A₂ isoform purified from this venom, myotoxin II, was also able to induce a systemic IL-6 release when injected into the footpad. Both venom and myotoxin induced local edema and a leukocyte infiltrate accumulating in the muscle and subdermal tissue within 6 h. The infiltrate consisted predominantly of neutrophils at 6 and 24 h, but at later times, mononuclear cells also appeared. The edema, leukocyte infiltration, and IL-6 responses did not depend on the hemorrhagic activity of venom, since all three effects were seen after injection of (1) preneutralized venom, devoid of hemorrhagic activity, and (2) purified myotoxin II. Circulating platelet numbers were significantly decreased 30 min after venom injection and returned to normal after 12 h. The venom also induced a rapid inversion in the ratio of neutrophils to lymphocytes in peripheral blood, which did not normalize until 12 h later. The present observations suggest that venom, besides its cytotoxic properties, induces early hematologic and immunologic alterations. These findings may be of relevance in future treatment modalities.     

A trade-off in replication in mosquito versus mammalian systems conferred by a point mutation in the NS4B protein of dengue virus type 4

An acceptable live-attenuated dengue virus vaccine candidate should have low potential for transmission by mosquitoes. We have identified and characterized a mutation in dengue virus type 4 (DEN4) that decreases the ability of the virus to infect mosquitoes. A panel of 1248 mutagenized virus clones generated previously by chemical mutagenesis was screened for decreased replication in mosquito C6/36 cells but efficient replication in simian Vero cells. One virus met these criteria and contained a single coding mutation: a C-to-U mutation at nucleotide 7129 resulting in a Pro-to-Leu change in amino acid 101 of the nonstructural 4B gene (NS4B P101L). This mutation results in decreased replication in C6/36 cells relative to wild-type DEN4, decreased infectivity for mosquitoes, enhanced replication in Vero and human HuH-7 cells, and enhanced replication in SCID mice implanted with HuH-7 cells (SCID-HuH-7 mice). A recombinant DEN4 virus (rDEN4) bearing this mutation exhibited the same set of phenotypes. Addition of the NS4B P101L mutation to rDEN4 bearing a 30 nucleotide deletion (Delta30) decreased the ability of the double-mutant virus to infect mosquitoes but increased its ability to replicate in SCID-HuH-7 mice. Although the NS4B P101L mutation decreases infectivity of DEN4 for mosquitoes, its ability to enhance replication in SCID-HuH-7 mice suggests that it might not be advantageous to include this specific mutation in an rDEN4 vaccine. The opposing effects of the NS4B P101L mutation in mosquito and vertebrate systems suggest that the NS4B protein is involved in maintaining the balance between efficient replication in the mosquito vector and the human host.     

Adoptive transfer of protection against Trypanosoma cruzi with lymphocytes and macrophages.

Female C57BL/6J mice were infected with Trypanosoma cruzi and subsequently given macrophages or lymphocytes from syngeneic donors which had recovered from the acute infection. Mice which received immune peritoneal macrophages, splenic lymphocytes, or lymph node lymphocytes developed lower mean parasitemias and cumulative mortalities than did recipients of nonimmune cells. Neither peritoneal lymphocytes nor splenic macrophages were protective, however. These studies indicate that splenic and lymph node lymphocytes are effective in transferring protection against T. cruzi, whereas the macrophage is somewhat less effective.     

Detectability in computed tomographic images.

The detection limitations inherent in statistically limited computed tomographic (CT) images are described through the application of signal detection theory. The detectability of large-area, low-contrast objects is shown to be chiefly dependent upon the low-frequency content of the noise power spectral density. For projection data containg uncorrelated noise, the resulting ramplike, low-frequency behavior of the noise power spectrum of CT reconstructions may be conveniently characterized by the number of noise-equivalent x-ray quanta (NEQ) detected in the projection measurements. The NEQ for a given image may be determined either from a measurement of the noise power spectrum or from the noise granularity computed with an appropriate weighting function. A measure of the efficiency of scanner dose utilization is proposed which compares the average dose to that required by an ideal scanner to obtain the same NEQ.     

A new PAX6 mutation in familial aniridia.

Aniridia (lack of iris) is caused by loss of function mutations in one copy of the PAX6 gene. Here we present a new PAX6 splice mutation in a family with autosomal dominant aniridia. The mutation is a single nucleotide change which, although occurring within an exon, affects the splice junction consensus and results in skipping of that exon.