Intestinal tuberculosis: a diagnostic challenge.

OBJECTIVE: To describe characteristics, presentation, time to diagnosis and diagnostic findings of patients with intestinal tuberculosis (ITB) in a low-burden country. METHOD: Retrospective study of 61 consecutive ITB patients diagnosed between 2008 and 2014 at a large East London hospital. RESULTS: Forty of sixty-one patients were male. Mean age was 34.6 years. 93% of patients were born abroad, mostly from TB-endemic areas (Indian subcontinent: 88%, Africa: 9%). 25% had concomitant pulmonary TB. Median time from symptom onset to ITB diagnosis was 13 weeks (IQR 3-26 weeks). Ten patients were initially treated for IBD, although patients had ITB. The main sites of ITB involvement were the ileocaecum (44%) or small bowel (34%). Five patients had isolated perianal disease. Colonoscopy confirmed a diagnosis of ITB in 77% of those performed. 42 of 61 patients had a diagnosis of ITB confirmed on positive histology and/or microbiology. CONCLUSION: Diagnosis of ITB is often delayed, which may result in significant morbidity. ITB should be excluded in patients with abdominal complaints who come from TB-endemic areas to establish prompt diagnosis and treatment. Diagnosis is challenging but aided by axial imaging, colonoscopy and tissue biopsy for TB culture and histology.

Airway mucosal inflammation in COPD is similar in smokers and ex-smokers: a pooled analysis.

Bronchial biopsy specimens from chronic obstructive pulmonary disease (COPD) patients demonstrate increased numbers of CD8+ T-lymphocytes, macrophages and, in some studies, neutrophils and eosinophils. Smoking cessation affects the rate of forced expiratory volume in one second (FEV(1)) decline in COPD, but the effect on inflammation is uncertain. Bronchial biopsy inflammatory cell counts were compared in current and ex-smokers with COPD. A pooled analysis of subepithelial inflammatory cell count data from three bronchial biopsy studies that included COPD patients who were either current or ex-smokers was performed. Cell count data from 101 subjects, 65 current smokers and 36 ex-smokers, were analysed for the following cell types: CD4+ and CD8+ T-lymphocytes, CD68+ (monocytes/macrophages), neutrophil elastase+ (neutrophils), EG2+ (eosinophils), mast cell tryptase+ and cells mRNA-positive for tumour necrosis factor-alpha. Current smokers and ex-smokers were similar in terms of lung function, as measured by FEV(1) (% predicted), forced vital capacity (FVC) and FEV(1)/FVC. The results demonstrate that there were no significant differences between smokers and ex-smokers in the numbers of any of the inflammatory cell types or markers analysed. It is concluded that, in established chronic obstructive pulmonary disease, the bronchial mucosal inflammatory cell infiltrate is similar in ex-smokers and those that continue to smoke.

Direct binding of estradiol enhances Slack (sequence like a calcium-activated potassium channel) channels' activity.

17Beta-estradiol (E2) is a major neuroregulator, exerting both genomic and non-genomic actions. E2 regulation of Slack (sequence like a calcium-activated potassium channel) potassium channels has not been identified in the CNS. We demonstrate E2-induced activation of Slack channels, which display a unitary conductance of about 60 pS, are inhibited by intracellular calcium, and are abundantly expressed in the nervous system. In lipid bilayers derived from rat cortical neuronal membranes, E2 increases Slack open probability and appears to decrease channel inactivation. Additionally, E2 binds to the Slack channel and activates outward currents in human embryonic kidney-293 cells that express Slack channels but not classical estrogen receptors (i.e. ERalpha or ERbeta). Neither E2-induced activation nor the binding intensity of E2 to the Slack channel is blocked by tamoxifen, an ER antagonist/agonist. Thus, E2 activates a potassium channel, Slack, through a non-traditional membrane binding site, adding to known non-genomic mechanisms by which E2 exerts pharmacological and toxicological effects in the CNS.

CNS stem and progenitor cell differentiation into functional neuronal circuits in three-dimensional collagen gels.

The mammalian central nervous system (CNS) has little capacity for self-repair after injury, and neurons are not capable of proliferating. Therefore, neural tissue engineering that combines neural stem and progenitor cells and biologically derived polymer scaffolds may revolutionize the medical approach to the treatment of damaged CNS tissues. Neural stem and progenitor cells isolated from embryonic rat cortical or subcortical neuroepithelium were dispersed within type I collagen, and the cell-collagen constructs were cultured in serum-free medium containing basic fibroblast growth factor. The collagen-entrapped stem and progenitors actively expanded and efficiently generated neurons, which developed neuronal polarity, neurotransmitters, ion channels/receptors, and excitability. Ca2+ imaging showed that differentiation from BrdU+/TuJ1- to BrdU-/TuJ1+ cells was accompanied by a shift in expression of functional receptors for neurotransmitters from cholinergic and purinergic to predominantly GABAergic and glutamatergic. Spontaneous postsynaptic currents were recorded by patch-clamping from precursor cell-derived neurons and these currents were partially blocked by 10-microM bicuculline, and completely blocked by additional 10 microM of the kainate receptor antagonist CNQX, indicating an appearance of both GABAergic and glutamatergic synaptic activities. Staining with endocytotic marker FM1-43 demonstrated active synaptic vesicle recycling occurring among collagen-entrapped neurons. These results show that neural stem and progenitor cells cultured in 3D collagen gels recapitulate CNS stem cell development; this is the first demonstration of CNS stem and progenitor cell-derived functional synapse and neuronal network formation in a 3D matrix. The proliferative capacity and neuronal differentiating potential of neural progenitors in 3D collagen gels suggest their potential use in attempts to promote neuronal regeneration in vivo.

A comparison of two reading interventions for children with reading disabilities.

This study compared the effectiveness of two reading interventions in a public school setting. Forty-five second-grade children with reading disabilities were randomly assigned to a 6-week phonological awareness, word analogy, or math-training program. The two reading interventions differed from each other in (a) the unit of word analysis (phoneme versus onset-rime), (b) the approach to intervention (contextualized versus decontextualized), and (c) the primary domain of reading instruction (oral versus written language). Results indicate that children in both reading programs achieved significant gains in beginning reading skills, learning the specific skills taught in their respective programs, and applying what they had learned to uninstructed material on several transfer-of-learning measures, in comparison to children in the control group. For children in both reading intervention groups, the most significant mediator of growth in oral reading fluency was a child's initial level of word identification skill. Implications of these findings are that systematic, high quality reading intervention can occur in a small group, public school setting and that there are several different paths to the remediation of children with reading disabilities.

Amyotrophic lateral sclerosis: serum factors enhance spontaneous and evoked transmitter release at the neuromuscular junction.

Sera from 30 patients with sporadic amyotrophic lateral sclerosis (ALS) were tested to determine their effects at the neuromuscular junction. Spontaneous transmitter release was significantly increased, as evidenced by a 151% increase in MEPP frequency, by sera from 16 ALS patients. In addition, 16 patients' sera elevated EEP quantal content by an average of 89%. Eleven sera produced both effects. There was no consistent change in MEPP amplitude or time course, indicating the absence of a humoral effect on postjunctional ACh receptors or endplate membrane function. These results suggest that a portion of the sporadic ALS patient population possess serum factors that can alter presynaptic function of the motor nerve terminal. Evidence from the present experiments indicates that alterations at the neuromuscular junction are a result of a combination of increased Ca2+ influx into the cell and an independent increase in intracellular calcium concentration.

Effect of pranlukast, an oral leukotriene receptor antagonist, on leukotriene D4 (LTD4) challenge in normal volunteers.

BACKGROUND: There is increasing evidence to show that leukotrienes are important mediators in asthma. Leukotriene receptor antagonists protect against antigen and exercise challenges in patients with chronic asthma. A study was undertaken to investigate the activity of the leukotriene receptor antagonist pranlukast (SB 205312, ONO-1078) in blocking bronchoconstriction induced by leukotriene D4 (LTD4) inhalation. The selectivity of pranlukast was evaluated using histamine challenge. METHODS: Pranlukast, 450 mg twice daily, was given to eight healthy non-smoking men for five days in a randomised, double blind, placebo controlled, crossover study. The specific airways conductance (sGaw) was measured before and after bronchial provocation with inhaled LTD4 at 3.5 hours after the first dose and at 3.5 and 9.5 hours after the last dose of pranlukast on the morning of day 5. The concentration of LTD4 required to produce a fall in sGaw of 35% (PC35) was calculated. Subjects also underwent a histamine challenge 3.5 hours after a single dose of pranlukast, 450 mg, or placebo. RESULTS: A single dose of pranlukast produced a 10.6 fold increase in PC35sGaw (95% confidence interval (CI) 4.4 to 25.5; p < 0.001) for LTD4 at 3.5 hours after dosing compared with placebo. Three and a half hours after the morning dose of pranlukast on day 5 the PC35sGaw for LTD4 was increased 25.9 fold (95% CI 10.8 to 62.2; p < 0.001) and was still increased sevenfold (95% CI 2.9 to 16.7; p < 0.001) relative to placebo 9.5 hours after administration of the morning dose. No significant differences were noted for the PC35sGaw to histamine for pranlukast compared with placebo. CONCLUSIONS: This study shows that pranlukast is a potent and selective LTD4 receptor antagonist in humans which blocks LTD4 challenge after initial and repeated administration when given twice daily for five days.

Inflammation in bronchial biopsies of subjects with chronic bronchitis: inverse relationship of CD8+ T lymphocytes with FEV1.

In order to determine whether the airway inflammatory cells of chronic obstructive pulmonary disease (COPD) are different from those seen in asthma, we have studied a subepithelial zone, 100 microns deep to the epithelial reticular basement membrane in bronchial biopsies taken from five normal nonsmoking subjects without chronic bronchitis or asthma (FEV1 percentage of predicted [mean +/- SD] 105.7 +/- 25.3), 11 subjects with chronic bronchitis alone (FEV1 percentage of predicted 98.5 +/- 12.9), and 13 subjects with chronic bronchitis in whom there was also evidence of airflow limitation (i.e., COPD; FEV1 percentage of predicted 59.7 +/- 10.0). Using immunohistochemical markers, we counted distinct types of inflammatory cell and expressed them as [median and range] per mm basement membrane. When there was airflow limitation we found significantly increased numbers of CD3+ T lymphocytes (COPD 22.3 [2.6 to 68.2] versus normal 3.7 [1.5 to 16.3]; p < 0.05), an increased number of CD8+ cells (COPD 19.3 [1.8 to 45.5] versus normal 2.3 [0.9 to 4.2]; p < 0.01), and increased expression of HLA-DR (COPD versus normal; p = 0.01). There was also an increased number of CD68+ cells (i.e., macrophages) (COPD 7.4 [0.4 to 16.9] versus normal 0.7 [0 to 2.6]; p < 0.01; COPD versus chronic bronchitis alone 2.7 [0 to 12.8]; p < 0.05). There were no significant differences between the groups in the numbers of subepithelial neutrophils, mast cells, eosinophils or B lymphocytes. There were weak but significant negative associations between the CD8+ T-cell subset (r = -0.42), neutrophils (r = -0.46), and eosinophils (r = -0.53) and FEV1 percentage of predicted in all the chronic bronchitic smokers (p < 0.05). The data confirm the involvement of subepithelial T lymphocytes and macrophages in smoking-induced airflow limitation and provide novel data which support the view that COPD is distinct from asthma with respect to the predominance of the CD8+ T-cell subset in this smoking-related condition.

Inhibition of calcium currents and exocytosis by Lambert-Eaton syndrome antibodies in human lung cancer cells.

1. Human small-cell lung cancer (SCLC) cells are believed to express the antigens responsible for the production of pathological antibodies in the Lambert-Eaton syndrome (LES), a Ca2+ channel disorder in which quantal transmitter release from the motor nerve terminal is impaired. Whole-cell patch-clamp techniques were used to study the voltage-dependent Ca2+ channels expressed by H146 SCLC cells and the effects of LES antibodies on these channels. The types of Ca2+ channels were determined using biophysical properties and pharmacological sensitivity to several antagonists. 2. Whole-cell Ca2+ currents (ICa) in SCLC cells are sensitive to the dihydropyridine (DHP) nicardipine, omega-conotoxin GVIA (omega-CgTX GVIA) and omega-agatoxin IVA (omega-AgTX IVA). Nicardipine at 100 nM and 10 microM reduced ICa by 35 and 45% (n = 38 cells), respectively, while omega-CgTX GVIA (1 microM) inhibited ICa by 32% (n = 31). Application of omega-AgTX IVA at 50 and 100 nM to the cancer cells decreased ICa by 41 and 42%, respectively (n = 22). 3. Measurement of cell membrane capacitance (Cm) revealed that Ca(2+)-dependent exocytosis underlies the secretory activity of SCLC cells. Exocytosis, when induced by step depolarizing pulses and measured by increases in Cm, was markedly inhibited by nicardipine (10 microM) and omega-AgTX IVA (100 nM). In contrast, omega-CgTX GVIA (1 microM) was not as effective in altering increases in Cm. 4. From negative (-80 mV) and depolarized (-40 mV) holding potentials, both peak and plateau ICa were inhibited by the presence of LES antibodies (1 mg ml-1 IgG). LES serum also reduced depolarization-induced increases in Cm by 48% (n = 15). 5. To determine whether the LES antibodies are downregulating a specific type(s) of Ca2+ channel, nicardipine (10 microM), omega-CgTX GVIA (1 microM) or omega-AgTX IVA (100 nM) was applied to tumour cells that had been previously exposed to LES serum for 24 h. The most pronounced change was that omega-AgTX IVA was 38-84% less effective at reducing ICa after the IgG treatment. The effectiveness of nicardipine was diminished by 18% after incubation with the LES antibodies, whereas the omega-CgTX GVIA was seen to be more effective. These results suggest that LES IgG downregulates P-type Ca2+ channels and, possibly, to a lesser extent L-type channels. 6. In view of recent evidence that P-type Ca2+ channels mediate cholinergic transmitter release at the mammalian neuromuscular junction (NMJ), the expression of P-type Ca2+ channels in the SCLC cells and the reactivity of LES IgG with these channels support the hypothesis that P-type Ca2+ channels in these cancer cells may trigger the autoantibody production in this disorder. The antibodies so produced are implicated in the functional impairment of the Ca2+ channels characteristic of LES.

A computer-based system for the measurement of membrane capacitance to monitor exocytosis in secretory cells.

A software-based system for real-time measurement of membrane capacitance was developed for monitoring exocytosis in a single cell. The IBM PC compatible software, linked to an interface box, provides capacitance measurement capabilities to a patch-clamp setup utilizing a Labmaster data acquisition board. In order to measure the time-dependent changes in membrane capacitance, the system uses a software-based phase detector which isolates capacitive membrane current from a cell subject to sinusoidal voltage stimulation. The sampling program can monitor changes in membrane capacitance in response to depolarizing voltage steps, intracellular calcium stimulation, or drug application. A separate analysis program was constructed to implement the quantitative analysis, plotting and exporting of the capacitance, conductance and current data acquired by the sampling program.

A computer program for the study of synaptic transmission at the neuromuscular junction.

The study of synaptic biopotentials at the neuromuscular junction has proven to be an invaluable tool in understanding the pre- and post-synaptic mechanisms underlying chemical synaptic transmission and exploring the pathogenesis of neuromuscular disorders. Computer technology has become an important part of these experiments as it increases accuracy and automates the process, allowing more data to be collected and quantitatively analysed. This paper presents a computer program for IBM PC compatible computers capable of recording and analysing the spontaneously occurring miniature end-plate potentials (MEPPs), neurally evoked end-plate potentials (EPPs) and muscle fiber action potentials (APs). The program performs its basic analysis immediately following the sampling of biopotentials from a muscle fiber, thus providing the user with immediate feedback on the status of the experiment; 45 parameters of neuromuscular transmission are computed on-line and displayed on the monitor along with the signal-averaged waveforms. After a series of fibers has been recorded, the program will compile the data into summary tables. The program is designed to provide an experimenter with an easy-to-use, comprehensive method of performing electrophysiological measurements and analysis at the neuromuscular junction.

Craniomandibular/temporomandibular/cervical implications of a forced hyper-extension/hyper-flexion episode (i.e., whiplash).

Clinicians now appreciate the full chain of tissue damage of the interconnecting muscles, tendons, ligaments and fascia: this compromised linkage from the skull through the suboccipital musculature to the cervical spine and anterior/posterior cervical muscles, from the check-rein ligaments and muscles extending from the skull and maxilla to the mandible, from the suprahyoid musculature connecting the mandible through to the hyoid bone, from the hyoid through the infrahyoid to the supporting shoulder girdle, all contribute to damaged interconnecting matrices. Consequently, unresolved tissue damage in any of this linkage becomes mutually provocative during function to any part of the linkage. Diagnostics, therefore, must necessarily include examination of this total linkage; similarly, eventual treatment protocols must resolve tissue damage in all of this interconnecting linkage if treatment is to be successful beyond palliative applications. The influence of an angular component in any such trauma must be emphasized; force vectors then attack the craniomandibular/temporo-mandibular/cervical complex where it is most vulnerable, at the most restrictive parameters of functional mobility. Thus, the angular component brings an exponential increase in tissue damage potential to all of the craniomandibular/temporomandibular/cervical complex. Finally, if we are to treat the occlusion of these victims intelligently, we must understand the effects of this trauma on the 'whole body', and not just focus singularly on the restoration or the malocclusion or the TMJoint problem. There are lessons to be learned from this acute trauma which provide valuable insights into the diagnosis of chronic pain patients. If these victims do not come to us for help immediately following the acute trauma episode, but arrive in our offices months or years later, our clinical examinations must include head and neck mobility or functional restrictions, observations of the patients' gait, other residual postural deficits, et al. Failure to implement these observations in our clinical examination will ultimately compromise our treatment success. As a final note, an axiom to bear in mind when studying the function of the musculoskeletal system is that when muscle is put into an adversarial relationship with bone, muscle always wins! Example #1: Compare a lateral cervical radiograph taken on the day of a rearend collision to one completed five months later; if tissue damage is unresolved, there will inevitably be a loss of lordotic curve of the cervical spine with a kyphosis at C-4, C-5!; Example #2: If a patient has developed a parafunctional habit like bruxism using pathological forces over a long period of time, the clinician will inevitably observe antigonial notching on the mandible at the masseter insertion! Although at first glance integrating these precepts into our examination protocols may appear to complicate matters, it actually makes our tasks easier and allows us to provide more comprehensive services for our patients. Those of us who are "functionally aware", will always be three steps ahead of the field in functional orthopedics or functional orthodontics. Let me conclude with a premise I learned many years ago from a very learned Osteopath, Dr. John Harakal, of Fort Worth, Texas: "If, as a clinician, you are able to put the body into a position to heal, it will heal itself."

Construction and use of integrative vectors to express foreign genes in mycobacteria.

We have constructed a mycobacterial integrative vector by placing two copies of the insertion sequence IS900 flanking a kanamycin-resistance gene into a 'suicide' vector unable to replicate in mycobacteria. The Mycobacterium leprae gene encoding the M. leprae 18 kDa protein was cloned between the two copies of IS900 to provide expression signals. Constructs were introduced into Mycobacterium species smegmatis, vaccae and bovis BCG by electroporation and selection for kanamycin resistance. The expression of the 18 kDa gene was analysed by Western blotting. Integration of the vector into the M. smegmatis chromosome was analysed by Southern blotting. One to five copies of the vector were detected in each transformant. The SIV gag p27 gene and the foot-and-mouth disease virus VP1 140-160 epitope were successfully cloned into the 18 kDa gene and expression in M. smegmatis was obtained.