Investigation of the regulation of transcriptional changes in Ancylostoma caninum larvae following serum activation, with a focus on the insulin-like signalling pathway.

The exit from dauer in the free-living nematode Caenorhabditis elegans is under the control of a single amphidial neuron (ASJ) of the insulin-like signalling pathway. Mutations of this pathway have the ability to suppress entry into the dauer stage. It has been postulated that insulin-like signalling plays a significant role in the response to serum stimulation in vitro of the third-stage larvae (L3s) of the canine hookworm Ancylostoma caninum. To test for the possible involvement of the insulin-like signalling cascade in the response to serum stimulation, the effects of two signalling stimulants (8-bromo cGMP and arecoline) and four inhibitors, namely 4,7-phenanthroline, phosphoinositide-3 kinase (PI3K), Akt inhibitor IV and rapamycin on feeding and on levels of selected activation-associated mRNAs in serum-stimulated L3s were explored. L3s of A. caninum were pre-incubated with or without the appropriate inhibitor/agonist. Following serum-stimulation, the feeding activity was assessed. The transcription levels of a number of activation-associated mRNAs linked to particular expressed sequence tags (ESTs) were investigated by reverse transcription, real-time PCR (rtPCR). The treatment of worms with 4,7-phenanthroline completely suppressed feeding and significantly reduced the differential levels of most activation-associated mRNAs, whereas the treatment with cGMP resulted in the resumption of feeding in almost 85% of the L3s and yielded a specific transcriptional profile consistent with that following serum stimulation. The treatment of L3s with arecoline resulted in the resumption of feeding in approximately 85% of L3s, but did not result in a transcriptomic profile consistent with activation. A complete reduction in feeding was recorded in the presence of the PI3K inhibitor LY294002 (1mM) and resulted in a pronounced dampening of differential transcription in response to serum stimulation for the molecules examined. Akt inhibitor IV resulted in a approximately 70% reduction in feeding but had almost no effect on the level of any of the activation-associated mRNAs studied. Rapamycin was shown to have a weak effect on feeding, and several of the mRNAs studied exhibited greater than expected transcription following treatment. The complexities of activation-associated transcription could not be addressed using the current approach. A larger number of mRNAs needs to be investigated in order to predict or identify regulatory mechanisms proposed to function in the insulin-like signalling pathway in A. caninum.

Transcriptional changes in the hookworm, Ancylostoma caninum, during the transition from a free-living to a parasitic larva.

BACKGROUND: Third-stage larvae (L3) of the canine hookworm, Ancylostoma caninum, undergo arrested development preceding transmission to a host. Many of the mRNAs up-regulated at this stage are likely to encode proteins that facilitate the transition from a free-living to a parasitic larva. The initial phase of mammalian host invasion by A. caninum L3 (herein termed "activation") can be mimicked in vitro by culturing L3 in serum-containing medium. METHODOLOGY/PRINCIPAL FINDINGS: The mRNAs differentially transcribed between activated and non-activated L3 were identified by suppression subtractive hybridisation (SSH). The analysis of these mRNAs on a custom oligonucleotide microarray printed with the SSH expressed sequence tags (ESTs) and publicly available A. caninum ESTs (non-subtracted) yielded 602 differentially expressed mRNAs, of which the most highly represented sequences encoded members of the pathogenesis-related protein (PRP) superfamily and proteases. Comparison of these A. caninum mRNAs with those of Caenorhabditis elegans larvae exiting from developmental (dauer) arrest demonstrated unexpectedly large differences in gene ontology profiles. C. elegans dauer exiting L3 up-regulated expression of mostly intracellular molecules involved in growth and development. Such mRNAs are virtually absent from activated hookworm larvae, and instead are over-represented by mRNAs encoding extracellular proteins with putative roles in host-parasite interactions. CONCLUSIONS/SIGNIFICANCE: Although this should not invalidate C. elegans dauer exit as a model for hookworm activation, it highlights the limitations of this free-living nematode as a model organism for the transition of nematode larvae from a free-living to a parasitic state.

Hookworm aspartic protease, Na-APR-2, cleaves human hemoglobin and serum proteins in a host-specific fashion.

Hookworms are voracious blood-feeders. The cloning and functional expression of an aspartic protease, Na-APR-2, from the human hookworm Necator americanus are described here. Na-APR-2 is more similar to a family of nematode-specific, aspartic proteases than it is to cathepsin D or pepsin, and the term "nemepsins" for members of this family of nematode-specific hydrolases is proposed. Na-apr-2 mRNA was detected in blood-feeding, developmental stages only of N. americanus, and the protease was expressed in the intestinal lumen, amphids, and excretory glands. Recombinant Na-APR-2 cleaved human hemoglobin (Hb) and serum proteins almost twice as efficiently as the orthologous substrates from the nonpermissive dog host. Moreover, only 25% of the Na-APR-2 cleavage sites within human Hb were shared with those generated by the related N. americanus cathepsin D, Na-APR-1. Antiserum against Na-APR-2 inhibited migration of 50% of third-stage N. americanus larvae through skin, which suggests that aspartic proteases might be effective vaccines against human hookworm disease.

Hemodynamic assessment of local anesthetic administration by laser Doppler flowmetry.

OBJECTIVE: The hemodynamic effects of local anesthetic administration with and without a vasoconstrictor were compared by using laser Doppler flowmetry. STUDY DESIGN: Seventeen people participated in a single study session in which they were given 2 intraoral injections. The injections, which were administered in random order, consisted of 1.8 mL lidocaine (2%) with epinephrine (1:100,000) and mepivacaine (3%). Hemodynamic parameters consisting of blood pressure, heart rate, and laser Doppler flowmetry were reordered at regular intervals. RESULTS: The laser Doppler flowmeter detected changes in the peripheral perfusion of the finger that were not detected by changes in blood pressure and heart rate. The greatest change was associated with anxiety and occurred just before the injection. The inclusion of epinephrine in the local anesthetic resulted in a persistence of these changes. CONCLUSION: This investigation has confirmed the sensitivity of laser Doppler flowmetry as an investigational tool for assessing hemodynamic changes associated with anxiety and the administration of local anesthesia.

Disruption of the ATM gene in breast cancer.

Mutations in the ATM gene, which maps to 11q22-23, cause the multisystem recessive syndrome ataxia-telangiectasia (AT). Breast cancer has been reported in AT patients and carriers. Sporadic breast cancer is associated with loss of heterozygosity at or in the region of ATM and chromosomal abnormalities involving 11q23. We have investigated the chromosomes, nuclei and released chromatin fibers from nine primary breast carcinoma and eight cell lines by fluorescence in situ hybridization with four fluorochrome-labeled cosmids spanning the ATM gene. The ATM gene was disrupted in one primary breast carcinoma and in the cell lines MDA-MB-231 and MCF-7. The role of these aberrations in breast carcinomas, which may lead to gene dosage or dominant negative effects on gene function, requires further investigation.

The radioulnar ratio: a new method of quantifying distal radioulnar joint subluxation.

Computed tomography was used to image the distal radioulnar joint (DRUJ) for instability. Four methods were used to quantify subluxation of the DRUJ: the Mino criteria, the epicenter method, the congruency method, and a new method called the radioulnar ratio (RUR). Validity of the various methods was evaluated in clinical and laboratory situations. Rheumatoid patients with symptomatic DRUJ pathology had significantly more abnormal RUR values (100% vs 73% [epicenter method] and 88% [Mino criteria]). The RUR detected instability sooner in a progressive laboratory-induced instability model. The intraobserver and interobserver reliability of the RUR was high, with intraclass correlation coefficients of 0.89 and 0.87, respectively. The RUR demonstrated superior performance in the diagnosis of DRUJ subluxation.

Preliminary report on a method of human papillomavirus DNA extraction from verruca plantaris for polymerase chain reaction studies.

This preliminary report on the Chelex-based extraction method of HPV DNA from verruca plantaris is the first step in a series of studies on verruca plantaris undertaken, jointly, by the Departments of Podiatric Medicine and Microbiology at Des Moines University-Osteopathic Medical Center, Des Moines, Iowa. Current projects include the development of new primers for PCR studies of HPV, exploration of different viral DNA extraction methods, and surveys concerning treatment, success of therapies, and the epidemiology of verruca plantaris.

Treatment of caliceal diverticular stones: transdiverticular percutaneous nephrolithotomy with creation of a neoinfundibulum.

PURPOSE: To evaluate transdiverticular percutaneous nephrolithotomy (TDPN) with creation of a neoinfundibulum in the treatment of caliceal diverticular stones. PATIENTS AND METHODS: Between 1990 and 1998, 18 patients with symptomatic calculi in caliceal diverticula underwent TDPN. Transdiverticular puncture into the renal collecting system with creation of a neoinfundibulum was used, eliminating the need for prolonged probing with a wire for the neck of the diverticulum. Eight diverticula were upper polar, six were interpolar, and four were in the lower pole. Stones were endoscopically treated with use of Lithoclast (Electromedical Systems, Lausanne, Switzerland), graspers, ultrasound, or a combination of these methods. RESULTS: Sixteen kidneys (89%) were rendered stone-free at discharge. Two kidneys (11%) were left with stone fragments of 5 mm or smaller. Hospital stay ranged from 3 to 15 days (average, 7 days). Procedure time ranged from 45 to 169 minutes (average, 87 minutes). One patient developed a left renal-pleural fistula, which closed 1 week after chest tube drainage. No other complications were encountered for an overall complication rate of 6%. CONCLUSION: TDPN is a safe and effective method for treating caliceal diverticular stones, with a complication rate comparable to other methods.

CT assessment of cerebral perfusion: experimental validation and initial clinical experience.

PURPOSE: To validate a dynamic single-section computed tomographic (CT) method to measure cerebral blood volume (CBV) and cerebral blood flow (CBF) by using a noncarotid artery as the input and to demonstrate the feasibility of this method in a pilot series of patients. MATERIALS AND METHODS: Twelve dynamic contrast material-enhanced CT studies were performed in beagles. CBV, CBF, and mean transit time (MTT) values were calculated by using an internal carotid artery (ICA) and a noncarotid artery as the input artery to the brain. Patient studies with use of the radial artery as the input were performed (a) repetitively in two patients after subarachnoid hemorrhage, (b) in a patient with a symptomatic ICA occlusion before and after the intravenous injection of 1 g of acetazolamide, and (c) in a patient with a malignant brain tumor. RESULTS: Linear regression analyses revealed highly significant correlations (P < .001) between CBV (r, 0.98; slope, 0.96), CBF (r, 0.89; slope, 0.87), and MTT (r, 0.80; slope, 0.76) values calculated with the ICA and the noncarotid inputs. The CT-derived patient data correlated well with ancillary clinical and neuroradiologic findings. CONCLUSION: Dynamic single-section CT scanning to measure CBV and CBF on the basis of a noncarotid input is a highly accessible and cost-effective blood flow measurement technique.

Acute acoustic trauma--its features and management.

The features and mechanism of Acute Acoustic Trauma (AAT) together with available treatments are discussed and the current guidelines in use by the Bundeswehr are also presented.

Sequence comparison of avian infectious bronchitis virus S1 glycoproteins of the Florida serotype and five variant isolates from Georgia and California.

The infectious bronchitis virus (IBV) spike glycoprotein S1 subunit is required to initiate infection and contains virus-neutralizing and serotype-specific epitope(s). Reported are the S1 gene nucleotide and predicted amino acid sequences for the Florida 18288 strain and isolates GA-92, CV-56b, CV-9437, CV-1686, and 1013. These sequences were compared with previously published gene sequences of IBV strains, and phylogenetic relationships are reported. The S1 amino acid sequence of Florida 18288 was 94.9% similar to the Connecticut strain, and GA-92 was 92.8% similar to the Arkansas 99 strain. S1 amino acid sequences of the California variants, CV-56b, CV-9437, and CV-1686, were 97.6-99.3% similar to one another and only 76.6%-76.8% similar to the Arkansas-type strains. Isolate 1013, also from California, was 84.0% similar to Ark DPI and 77.9% similar to CV-56b. When comparing 19 viruses isolated from the United States, sequence variations were observed between amino acids 55-96, 115-149, 255-309, and 378-395. Similar regions are reported to be involved in virus-neutralizing and/or serotype-specific epitopes. These data demonstrate that variant IBV strains continue to emerge, and unique variants may circulate among poultry in geographically isolated areas.

Treatment of xanthelasma palpebrarum with bichloracetic acid.

BACKGROUND: Although many treatment modalities have been described for xanthelasma palpebrarum, no single technique has emerged as dominant. OBJECTIVES: Our purpose was to review the various therapeutic modalities for xanthelasma and to assess the efficacy of topical bichloracetic acid. METHODS: Thirteen patients with 25 xanthelasma were treated with topical 100% bichloracetic acid. Efficacy was assessed over a follow-up period of 7 months to 10.5 years (average, 64 months). RESULTS: Eighty-five percent of patients experienced initial complete clearing, and 72% of their lesions have not required retreatment over an average period of 68 months. Recurrences responded well to repeat treatment. Eighty-three percent of recurrent or poorly responsive lesions were associated with high cholesterol. The most resistant patient had four-lid involvement. Excellent cosmetic results and high patient satisfaction were seen. CONCLUSIONS: Topical bichloracetic acid is a viable alternative to other modalities in the management of xanthelasma. Advantages include simplicity, cost-effectiveness, speed, safety, and efficacy.

Diagnostic pitfalls in sports related laryngeal injury.

The serious nature of laryngeal trauma sustained during sporting activity can be easily overlooked. Concomitant injury should not distract from the diagnosis of potential airway problems. The effects of head and neck trauma on the airway may be delayed. The assessment and management of a patient with suspected laryngeal injury should be carried out by experienced medical practitioners.

Complications of radiologically placed central venous ports and Hickman catheters in patients with AIDS.

OBJECTIVE: To determine any differences in the complications following radiologic placement of 2 devices--Hickman catheters and central venous ports--to permit long-term central venous access in patients with AIDS. PATIENTS AND METHODS: Thirty-eight patients with AIDS received 52 long-term central venous access catheters. Seventeen received 23 Hickman catheters and 27 received 29 central venous ports (hence, 6 patients received both). Complications rates were determined retrospectively from clinical records and radiologic studies. RESULTS: The only acute complications were pneumothoraces--3 in the patients receiving Hickman catheters and 2 in the patients receiving central venous ports. Infections developed in 12 of the patients receiving Hickman catheters a median of 53 and a mean of 96 days after placement. Infection developed in 8 of the patients receiving central venous ports a median of 125 days and a mean of 184 days after placement. This difference was statistically significant (p < 0.001). The most common organism in these infections was Staphylococcus aureus. The only other nonacute complication was 1 nonocclusive thrombosis of the superior vena cava, which occurred in a patient with a Hickman catheter 240 days after placement and resolved once the catheter was removed. CONCLUSION: Central venous ports are preferred over Hickman catheters for long-term central venous access in patients with AIDS.

Measurement of the scaphoid humpback deformity using longitudinal computed tomography: intra- and interobserver variability using various measurement techniques.

The intra- and interobserver variability of 3 techniques for measuring the humpback deformity of 37 scaphoids using longitudinal computed tomography was assessed. The 3 measuring techniques were the lateral intrascaphoid angle, the dorsal cortical angle, and the height-to-length ratio. The intraobserver reliability of the intrascaphoid angle was poor; the dorsal cortical angle was moderate to excellent, and the height-to-length ratio was excellent. The interobserver reliability of the intrascaphoid angle was poor to moderate, the dorsal cortical angle was moderate to excellent, and the height-to-length ratio was moderate to excellent. For all 3 observers, the intra- and interobserver reliability was the best for the height-to-length ratio and worst for the intrascaphoid angle. The height-to-length ratio is the most reproducible method of assessing the humpback deformity. Clinical correlation is required to establish whether the height-to-length ratio will be of value in predicting the outcome of fractures of the scaphoid.

Bronchogenic cyst: an unusual cause of lump in the neck.

Bronchogenic cysts are rare congenital benign lesions that are usually detected in the paediatric patient with symptoms of infection or compression on vital structures. They are rarely diagnosed in the adult population. We present a case of bronchogenic cyst presenting as a lump in the neck in an adult patient. Radiological imaging helped to diagnose this lesion accurately before any form of intervention. Complete surgical excision is the treatment of choice.