Mediastinal tumors: biopsy under US guidance

Percutaneous biopsies of mediastinal tumors were successfully performed under sonographic guidance in 14 of 21 patients. In 10 of 11 malignant lesions, malignancy was determined by means of cytologic and histologic examination of the specimens obtained. A histologic diagnosis was reached in seven patients with malignant mediastinal tumors, including all four cases of Hodgkin lymphoma. Mediastinal biopsy under sonographic guidance is a technically simple, rapid, and accurate procedure, but its application is limited to tumors of the anterior mediastinum.     

Intravenous digital subtraction renal angiography: use in screening for renovascular hypertension

Intravenous digital subtraction renal angiography (DSRA) has been compared with conventional angiography only in small, selected series of hypertensive patients. The authors prospectively examined with intravenous DSRA 94 patients at increased risk for renovascular hypertension and compared these studies with conventional angiography. A stenosis of at least one main renal artery was identified with intravenous DSRA in 22 patients and confirmed in 20 patients. No significant stenoses were seen with conventional angiography in any of the 64 patients in whom lesions were not seen with intravenous DSRA. Since inadequate DSRA studies were considered positive for renal artery stenosis, the sensitivity of intravenous DSRA was 100% (25 of 25); specificity, 93% (64 of 69); positive predictive value, 83% (25 of 30); and negative predictive value, 100% (64 of 64). The authors conclude that intravenous DSRA is a sensitive test for identifying stenosis of the main renal arteries and is appropriate to use as a screening test among patients at increased risk for renovascular hypertension.     

DNA melting analysis for detection of single nucleotide polymorphisms

BACKGROUND: Several methods for detection of single nucleotide polymorphisms (SNPs; e.g., denaturing gradient gel electrophoresis and denaturing HPLC) are indirectly based on the principle of differential melting of heteroduplex DNA. We present a method for detecting SNPs that is directly based on this principle. METHODS: We used a double-stranded DNA-specific fluorescent dye, SYBR Green I (SYBR) in an efficient system (PE 7700 Sequence Detector) in which DNA melting was controlled and monitored in a 96-well plate format. We measured the decrease in fluorescence intensity that accompanied DNA duplex denaturation, evaluating the effects of fragment length, dye concentration, DNA concentration, and sequence context using four naturally occurring polymorphisms (three SNPs and a single-base deletion/insertion). RESULTS: DNA melting analysis (DM) was used successfully for variant detection, and we also discovered two previously unknown SNPs by this approach. Concentrations of DNA amplicons were readily monitored by SYBR fluorescence, and DNA amplicon concentrations were highly reproducible, with a CV of 2.6%. We readily detected differences in the melting temperature between homoduplex and heteroduplex fragments 15-167 bp in length and differing by only a single nucleotide substitution. CONCLUSIONS: The efficiency and sensitivity of DMA make it highly suitable for the large-scale detection of sequence variants.     

Defects of T-cell effector function and post-thymic maturation in X-linked hyper-IgM syndrome

X-linked hyper-IgM syndrome (XHIM) results from mutations in the gene encoding for CD40 ligand (CD154). Patients with the syndrome suffer from infections with opportunistic pathogens such as Cryptosporidium and Pneumocystis carinii. In this study, we demonstrate that activated T cells from patients with XHIM produce markedly reduced levels of IFN-gamma, fail to induce antigen-presenting cells to synthesize IL-12, and induce greatly reduced levels of TNF-alpha. In addition, we show that the patients' circulating T lymphocytes of both the CD4(+) and CD8(+) subsets contain a markedly reduced antigen-primed population, as determined by CD45RO expression. Finally, we demonstrate that the defects in antigen priming are likely due to the lack of CD154 expression and insufficient costimulation of T cells by CD80/CD86 interactions. Taken together, this study offers a basis for the increased susceptibility of patients with XHIM to certain opportunistic infections.     

Cephalosporin-induced hypoprothrombinemia: possible role for thiol methylation of 1-methyltetrazole-5-thiol and 2-methyl-1,3,4-thiadiazole-5-thiol

Heterocyclic thiol metabolites of cephalosporin antibiotics may play an important role in the pathophysiology of hypoprothrombinemia and hemorrhage in patients treated with these drugs. A heterocyclic thiol metabolite of moxalactam, 1-methyltetrazole-5-thiol (MTT), inhibits the gamma carboxylation of glutamic acid that is required for the formation of active clotting factors. One possible pathway for the biotransformation of thiol compounds such as MTT is S-methylation catalyzed by either thiopurine methyltransferase (TPMT), a soluble enzyme, or by thiol methyltransferase, a microsomal enzyme. Therefore, MTT and 2-methyl-1,3,4-thiadiazole-5-thiol (MTD), a thiol "leaving group" structurally related to MTT that is present in cefazolin, were tested as possible substrates for S-methylation catalyzed by purified human kidney TPMT or by human liver microsomes, a source of thiol methyltransferase. MTT and MTD were methylated by both human kidney TPMT and human liver microsomes. The products of these reactions were shown by high-performance liquid chromatography to be S-methyl MTT and S-methyl MTD. Apparent Km constants for the methylation of MTT and MTD by TPMT were 0.26 and 0.068 mM, respectively. Apparent Km constants for the methylation of MTT and MTD by human liver microsomes were 0.60 and 0.20 mM, respectively. Maximal velocity (Vmax) values for the S-methylation of MTD catalyzed by TPMT and by human liver microsomes were 3.58- and 678-fold greater than were those for the thiol methylation of MTT. Finally, S-methyl derivatives of MTT and MTD were one to two orders of magnitude less potent as inhibitors of the in vitro gamma carboxylation of glutamic acid than were MTT and MTD themselves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of Human Helper T Cell Function In Vitro by d-Penicillamine and CuSO4

The effect of d-penicillamine (Pen) and mixtures of Pen and copper sulfate on the capacity of normal human peripheral blood mononuclear cells (PBM) to generate immunoglobulin-secreting cells (ISC) in response to the T-cell-dependent polyclonal B-cell activators pokeweed mitogen (PWM) and staphylococcal protein A (SPA) was examined. PBM obtained from normal individuals were incubated for 1-2 h at 37 degrees C with medium alone, Pen, CuSO(4), or a mixture of Pen and CuSO(4). After washing, the cells were incubated for 6-7 d with PWM or SPA and then, with a reverse hemolytic plaque assay, assayed for the number of ISC generated. Preincubation of PBM with either Pen (100 mug/ml) or CuSO(4) (2 mug/ml) did not alter the subsequent capacity of the cells to generate ISC in response to PWM or SPA. In contrast, responsiveness to both mitogens was nearly abolished when PBM were similarly preincubated with a mixture of Pen and CuSO(4). Inhibition of responsiveness could not be ascribed to cell death, carry-over of the inhibitors, or an alteration in the concentration of PWM or the length of incubation yielding maximum responses. Co-culture experiments demonstrated that Pen and CuSO(4) preincubation had not caused augmented suppressor cell function. Experiments in which PBM were separated into adherent and nonadherent populations indicated that Pen and CuSO(4) preincubation inhibited the responsiveness of the nonadherent cells but did not alter the accessory cell function of monocytes. To determine whether Pen and CuSO(4) preincubation effected T- or B-cell function, PBM were separated into B- and T-cell-enriched populations, individually preincubated with Pen and CuSO(4), and then co-cultured with PWM. The results indicated that Pen and CuSO(4) markedly inhibited helper T-cell function and had little effect on the capacity of B cells to generate ISC. The observation that in the presence of CuSO(4) Pen inhibits helper T-cell activity may, in part, explain the therapeutic efficacy of Pen in rheumatoid arthritis and especially the capacity of Pen therapy to decrease antiglobulin titers in treated patients.     

Activation of T lymphocytes by immobilized monoclonal antibodies to CD3. Regulatory influences of monoclonal antibodies to additional T cell surface determinants

The effect of soluble or immobilized MAb directed at various additional surface proteins on the proliferation of highly purified T4 cells induced by two immobilized MAb to CD3, OKT3 and 64.1, was examined. High density 64.1 stimulated nearly all T4 cells to enter and progress through the cell cycle. Maximal T4 cell proliferation required stimulation with immobilized 64.1 throughout the length of the incubation and was not effected by any of the additional soluble or immobilized MAb employed. In contrast, low density immobilized 64.1 and all densities of immobilized OKT3 employed stimulated a minority of the cells to enter the cell cycle and proliferate. Immobilized MAb directed at CD2, class I major histocompatibility complex (MHC) encoded gene products or CD11a (LFA-1) dramatically enhanced the response, whereas soluble MAb directed at these determinants did not. Both immobilized and soluble MAb directed at CD5 and CD28 (Tp44) enhanced responses, but they were less effective than immobilized MAb to CD2, LFA-1 or HLA-A,B,C. Soluble anti-CD4 MAb inhibited responses somewhat, whereas immobilized anti-CD4 enhanced responses. Costimulation was observed when MAb to CD3 and class I MHC molecules but not CD2, LFA-1 or CD4 were immobilized to separate surfaces. The data suggest that when anti-CD3 stimulation is suboptimal, responses can be enhanced by MAb to CD5 or CD28 (Tp44) or by immobilized MAb to CD4, CD2, CD11a (LFA-1), or class I MHC encoded gene products. Although crosslinking of CD4, CD2, or CD11a with CD3 may be necessary for costimulation, immobilized MAb to CD3 and class I MHC molecules appear to deliver independent signals that result in enhanced T4 cell activation and proliferation.