Use of PCR for Diagnosis of Post-Kala-Azar Dermal Leishmaniasis

Microscopy and PCR were compared for use in the diagnosis of post-kala-azar dermal leishmaniasis (PKDL) in 63 patients. Aspirates of lymph nodes (samples from 52 patients), skin (23 samples), and bone marrow (18 samples) were used. For 11 patients lymph node aspiration could be repeated 6 months after they recovered from PKDL. During active PKDL, PCR was positive for 42 of 52 (80.8%) lymph node aspirates and 19 of 23 (82.7%) skin aspirates, whereas microscopy was positive for only 9 of 52 (17.3%) lymph node aspirates and 7 of 23 (30.4%) skin aspirates. PCR was always positive when parasites were seen by microscopy. When the results obtained with lymph node and skin aspirates from the same patient (n = 16) were compared, there was complete agreement. Bone marrow samples were negative by microscopy and PCR for 16 patients and positive by both methods for 1 patient; for one sample only the PCR was positive. PCR confirmed the co-occurrence of visceral leishmaniasis and PKDL in one patient and confirmed the suspicion of this co-occurrence in the other patient. After recovery, no parasites were found by microscopy, but 2 of 11 (18.2%) samples were still positive by PCR. Thirty negative controls were all found to be PCR negative, and 15 positive controls were all PCR positive. Cross-reactions with Mycobacterium leprae could be ruled out. In conclusion, PCR with inguinal lymph node or skin aspirates is suitable for confirming the clinical diagnosis of PKDL. In some patients, lymph node aspirates are probably preferred because aspiration of material from the skin may leave scars.     

Function and structure of pressurized and perfused porcine carotid arteries: effects of in vitro balloon angioplasty

In this report we describe the application of an in vitro pressure-perfusion system for study of functional/structural changes after in vitro balloon dilation injury. Pig carotid arteries were perfused at P = 100 mm Hg and Q = 100 ml/min, balloon angioplastied (BA), and cultured under these hemodynamic conditions for 4 or 8 days (n = 5 BA and 6 controls for each time point). To assess endothelial function, outer diameter changes in response to bradykinin (BK) were measured daily. Remodeling was determined from the shift in pressure-passive diameter relation, as obtained after papaverine addition. Arterial samples were processed for histology. Control arteries showed spontaneous tone, BK-induced relaxation, and inward remodeling that was more pronounced at day 8 (ratio end-to-start passive diameter at P = 100 mm Hg, 0.69 +/- 0.04; P < 0.001) than at day 4 (0.85 +/- 0.03, P = 0.03). Intimal hyperplasia was detectable in these control vessels at day 8 with accumulation of alpha-smooth muscle actin-positive cells around the lumen. Angioplasty caused ruptures and dissections and abolished tone that returned after 5 days of perfusion along with BK-dependent relaxation. No significant inward remodeling or intimal hyperplasia was observed at day 8 after angioplasty. Thus, BA inhibits remodeling, which occurs after in vitro perfusion of conductance arteries.     

Amplified region of chromosome band 11q13 in breast and squamous cell carcinomas encompasses three CpG islands telomeric of FGF3, including the expressed gene EMS1

DNA markers that map within the karyotypically defined band q13 on human chromosome 11 are amplified in a subset of mammary and squamous cell carcinomas. It is assumed that the amplified DNA includes a critical gene (or genes) whose overexpression provides a selective force in the development of the tumor. To help identify such genes, we have begun to construct a physical map of CpG islands in the region, making use of a squamous cell carcinoma cell line (UMSCC2) in which the 11q13 region is amplified 11-fold. We previously described the proximal end of this amplicon and the order of markers extending ～800 kb centromeric of the FGF3 locus (formerly INT2). We now report the use of chromosome jumping techniques to define additional CpG islands that lie distal to FGF3. These map within the amplified region in UMSCC2 cells and the most telomeric corresponds to the EMS1 gene. The data imply that the amplified DNA in UMSCC2 cells extends for over 1,500 kb and includes at least 7 potential genes. EMS1 and CCND1 (formerly PRAD1), the best candidates for the key gene on the 11q13 amplicon, are ≥800 kb apart. © 1993 Wiley-Liss, Inc.     

Theophylline and fibrocystic breast disease

After literature reports linking fibrocystic breast disease (FBD) to methylxanthine ingestion, a pilot study was undertaken to investigate the possible contribution of theophylline to this effect. The major goal of this project was to measure the effect of theophylline therapy on FBD in asthmatic women. All women attending an allergy clinic or an obstetrics/gynecology clinic over a 9-month period were examined to clinically assess FBD and were asked to complete a detailed questionnaire covering health history, other risk factors, and drug and dietary methylxanthines. The sample included 62 asthmatic women, 66 allergic but not asthmatic women, and 72 nonallergic and nonasthmatic women. By use of the FBD clinical taxonomy with its 19-point scale going from 0 to 18 that was developed for this study, the three groups did not differ significantly in terms of mean severity of FBD. On analyzing the effect of each of the methylxanthines on FBD severity, there is clear evidence that total methylxanthines was a contributing factor in FBD severity with or without adjustment for relevant variables, such as age, menopause, pregnancies, and groups. Theophylline was significant only when adjustments were made for age, pregnancy, and menopause in contrast to caffeine that was only significant with no adjustments.     

Malaria vectors in the Bioko Island (Equatorial Guinea): estimation of vector dynamics and transmission intensities

The current study was performed on the Bioko Island (Equatorial Guinea) with the aim of establishing a rapid assessment technique for mapping malaria risk and measuring vector densities. Human bait collection, tent traps, light traps, indoor resting collection, and window exit traps were used to collect Anopheles gambiae s.s. and Anopheles funestus, the two anopheline species involved in malaria transmission in this island. Capture data were used to compare differences in the behavior and vectorial capacity of An. gambiae s.s. and An. funestus. Differences in the two species of mosquitoes were found in relation to the season and trapping methods used. Entomological inoculation rates (EIR) for Plasmodium falciparum were calculated using a polymerase chain reaction (PCR) test with individual anopheline mosquitoes from human bait collections in two villages during the dry and rainy seasons. P. falciparum sporozoites were detected from both dissected heads/thorax and abdomens of both species.     

In vivo magnetic resonance imaging explorative study of ectopic bone formation in the rat

In animal studies of tissue engineering of bone, histology remains the standard for assessing bone formation. As longitudinal studies with this method are feasible only at the cost of large numbers of animals, we looked for an alternative. Therefore, demineralized bone matrix (DBM) and inactivated demineralized bone matrix (iDBM) implants were subcutaneously implanted in a rat. At 1, 3, 5, and 7 weeks postimplantation soft X-ray and magnetic resonance imaging (MRI) were done to monitor bone formation in the implants. At 7 weeks, the animal was killed and the implants were retrieved for histology. Our results showed that in vivo MRI is well suited to assess bone formation larger than 0.5 mm in diameter and to monitor the complete three-dimensional shape of the newly formed bone noninvasively and longitudinally. The MRI results matched well with the histology results obtained at 7 weeks. In contrast, X-ray imaging appeared inappropriate to monitor the bone formation process in DBM.     

A chimeric T cell receptor with super-signaling properties

A key question yet to be resolved concerns the structure and function relationship of the TCR complex. How does antigen recognition by the TCR-alphabeta chains result in the activation of distinct signal transduction pathways by the CD3-gammadeltaepsilon/zeta complex? To investigate which part of the TCR-beta chain is involved in TCR signaling, we exchanged different domains of the constant regions of the TCR-beta chain with the corresponding TCR-gamma chain domains. We show here that hybridoma cells expressing a chimeric TCR-beta chain (betaIII) containing intracellular and transmembrane TCR-gamma amino acids, together with a wild-type TCR-alpha (alphawt) chain, were 10 times more sensitive to antigenic stimulation compared to cells expressing TCR-alphawt/betawt chains. This super-signaling phenotype of the betaIII chain was observed in two different TCRs. One specific for an alloantigen (I-A(bm12)) and one for an autoantigen (I-A(b)/MOG(35-55)). We found that this chimeric alphawt/betaIII TCR had normal association with CD3-gammadeltaepsilon and zeta chains. To investigate the effect of the chimeric betaIII chain in transgenic T cells, we made MOG(35-55)-specific TCR transgenic mice expressing either the alphawt/betawt or chimeric alphawt/betaIII TCR. Similar to what was observed in hybridoma cells, transgenic alphawt/betaIII T cells showed a super-signaling phenotype upon antigenic stimulation. Further studies may help us understand the effect of increased TCR signaling on autoimmunity and may lead to the identification of signaling molecules that can be targeted to stop the progression of autoimmune disorders such as multiple sclerosis.     

Simultaneous mapping of human papillomavirus integration sites and molecular karyotyping in short-term cultures of cervical carcinomas by using 49-color combined binary ratio labeling fluorescence in situ hybridization

Infection with high-risk type human papillomavirus (HPV) is a necessary causal factor in the pathogenesis of cervical carcinoma. In most invasive cervical cancers, HPV is integrated in the host cell genome, and additional genetic aberrations are observed among which are chromosomal aberrations. To analyze in detail such often complex chromosomal changes and simultaneously map HPV integration sites, we extended the multiplicity of the combined binary ratio labeling fluorescence in situ hybridization (COBRA-FISH) technique to 49 by inclusion of a large Stokes' shift fluorochrome as the third binary label. The technique allows mapping of the integrated HPV genome in the context of p- and q-arm COBRA-FISH, with a sensitivity of one copy of the HPV genome as tested for HPV 16 in SiHa cells. We investigated the molecular karyotypes and integration patterns of HPV types 16 and 18 in metaphase spreads from short-term cultures of primary cervical carcinomas (n=5). Of the tested cervical carcinomas, two contained integrated HPV at 8q24, one of which in addition harbored the integrated virus near a translocation breakpoint. Two carcinomas had integrated HPV at 17q21 through 23 in a morphologically normal chromosome 17. One carcinoma contained HPV at 1q42 in a morphologically normal chromosome 1. Our data illustrate the efficacy of 49-color COBRA-FISH to resolve complex karyotypes and simultaneously map specific sequences in metaphases obtained from short-term solid tumor cultures.     

Resident Enteric Bacteria Are Necessary for Development of Spontaneous Colitis and Immune System Activation in Interleukin-10-Deficient Mice

Mice with targeted deletion of the gene for interleukin-10 (IL-10) spontaneously develop enterocolitis when maintained in conventional conditions but develop only colitis when kept in specific-pathogen-free (SPF) environments. This study tested the hypothesis that enteric bacteria are necessary for the development of spontaneous colitis and immune system activation in IL-10-deficient mice. IL-10-deficient mice were maintained in either SPF conditions or germfree conditions or were populated with bacteria known to cause colitis in other rodent models. IL-10-deficient mice kept in SPF conditions developed colitis in all segments of the colon (cecum and proximal and distal colon). These mice exhibited immune system activation as evidenced by increased expression of CD44 on CD4⁺ T cells; increased mesenteric lymph node cell numbers; and increased production of immunoglobulin A (IgA), IgG1, and IL-12 p40 from colon fragment cultures. Mice populated with bacterial strains, including Bacteroides vulgatus, known to induce colitis in other rodent models had minimal colitis. Germfree IL-10-deficient mice had no evidence of colitis or immune system activation. We conclude therefore that resident enteric bacteria are necessary for the development of spontaneous colitis and immune system activation in IL-10-deficient mice.