Generation of human T lymphocytes from bone marrow CD34+ cells in vitro.

Analysis of the events that regulate development of red blood cells or granulocytes has led to therapies altering clinical conditions associated with anemia or neutropenia. The development of therapeutic approaches to target conditions associated with lymphopenia, such as AIDS, has been thwarted by limited techniques for studying T-lymphocyte development. We describe an in vitro system in which human bone marrow CD34+ cells proliferate, acquire the expression of the lymphoid-specific RAG-2 gene and a broad repertoire of rearranged T-cell receptor genes, develop the ability to produce T cell-specific interleukin-2 and achieve a range of T-cell immunophenotypes. The cells also become susceptible to infection with the T-lymphotropic strain of human immunodeficiency virus-1, HIV-1IIIB. This culture system induces human T lymphopoiesis and may permit further analysis of the events regulating human T-lineage differentiation. It provides a preclinical model for screening stem cell gene therapies directed toward AIDS.

Human immunodeficiency virus infection of eosinophils in human bone marrow cultures.

Normal human bone marrow, cultured in vitro with interleukin 5 to promote eosinophil production and maturation, was inoculated with cell-free isolates of human immunodeficiency virus type 1 (HIV-1). CD4 expression by eosinophil precursors, determined by immunocytochemistry, was found to be greatest early in their maturation with a rapid decline after 28 d in culture. Productive HIV infection of eosinophil precursors was detected 14 d after inoculation, by a combination of immunostaining for HIV-1 p24 and gp41/160 and in situ hybridization for viral RNA, together with assay of culture supernatants for p24 antigen and reverse transcriptase activity. Thus, eosinophils are susceptible to productive HIV-1 infection in vitro and may be an important reservoir for the virus in vivo.

Hysteresis of the ventricular paced QT interval in response to abrupt changes in pacing rate.

The rate of QT adaptation to abrupt changes in pacing rate was studied in seven patients with newly diagnosed complete heart block with a ventricular escape rate of less than 40 beats.min-1. Their median age was 70 (range 36-84) years, and none was taking any cardioactive medication known to affect the QT interval. From a baseline pacing rate of 50 or 110 beats.min-1 the ventricular rate was increased or decreased to a new level. The time taken for the ventricular paced QT interval to complete 90% of the change secondary to the change in rate was found to be 136(16) s (mean(SEM] when the rate was increasing and 189(25) s when the rate was decreasing (p less than 0.01). This time interval was independent of the magnitude of the rate change and the baseline heart rate from which the change occurred. Furthermore, the time course of QT adaptation was found to be exponential and was characterised by a time constant of 49.1(2.2) s when the rate was increasing and 60.4(2.0) s when the rate was decreasing (p less than 0.01). It is concluded that QT measurements in response to a change in pacing rate should take into account the time dependent nature of QT changes.

The production and characterisation of monoclonal antibodies to wheat gliadin peptides.

BALB/c mice maintained on a gluten-free diet were immunised with Frazer's Fraction III (FFIII, a peptic tryptic digest of wheat gluten that exacerbates coeliac disease) in order to overcome oral tolerance. A control group was maintained on normal diet. Serum antibody titres to FFIII were higher in the mice on a gluten-free diet (P less than 0.05). Three monoclonal antibodies to FFIII were produced from splenocytes obtained from mice maintained on a gluten-free diet. The antibodies were characterised by ELISA, immunodot assay and immunoblotting with prolamins from cereals toxic to coeliac patients (wheat, rye, barley, oats) and the non-toxic prolamins from maize and rice. The binding characteristics of the three antibodies to the cereal prolamins were different, implying that the antibodies recognise different cereal protein epitopes. Immunoblotting revealed FFIII to be comprised of antigenically dissimilar peptides.

Monoclonal antibody ELISA to quantitate wheat gliadin contamination of gluten-free foods.

A sensitive reproducible monoclonal antibody-based sandwich ELISA has been developed to measure the gliadin content of foods. Using the assay we have confirmed that nominally gluten-free products based on wheat starch contain trace amounts of gliadin. The level of gliadin contamination in these foods is sufficient to cause relapse of coeliac disease in specific patients. We have also examined the cross-reactivity of different cereal proteins in the assay and found good correlation with their known toxicity to patients with coeliac disease.

Energy requirements of the respiratory musculature in asthma.

Oxygen consumption and carbon dioxide production were measured in 29 asthmatic subjects. Minute ventilation was measured by a rib cage and abdomen-diaphragm displacement method. Expired gases were collected via a tight-fitting mask. Minute ventilation, oxygen consumption, and carbon dioxide production all increased when subjects inspired room air via a mouthpiece (when compared with a tight-fitting mask). By contrast, minute ventilation and carbon dioxide production both decreased when supplementary oxygen replaced room air via the tight-fitting mask (p less than 0.001). No consistent changes in either inspiratory work (estimated from measurement of pleural pressure during quiet breathing), airway resistance, or physiologic dead space could be seen to accompany changes in minute ventilation. It is concluded that the oxygen cost of breathing in asthma is substantial.

Review article: the use of biotherapeutic agents in the prevention and treatment of gastrointestinal disease.

There is presently a lack of well conducted clinical trials demonstrating any significant benefits of probiotics in humans. With the exception of diarrhoea due to rotavirus infection in children there is little evidence from randomized, double-blind, placebo-controlled studies that bacterial probiotics have a significant beneficial action in preventing diarrhoea of any cause. The yeast Saccharomyces boulardii has been shown to be of benefit in the prevention of antibiotic-associated diarrhoea but not in preventing infection with Clostridium difficile. S. boulardii may also be of benefit in preventing relapse of C. difficile infection. Because of the simplicity of in vitro systems and some animal models, beneficial characteristics of probiotics such as the ability of bacteria to bind to epithelial surfaces are not always transferable to humans. Thus any postulated benefit from consumption of probiotic bacteria should only be accepted as fact after testing in clinical studies. This review outlines our present knowledge of the mode of action of probiotics and presents the data from clinical trials on their use.

Timing of infiltration of T lymphocytes induced by gluten into the small intestine in coeliac disease.

Jejunal biopsy was performed on control subjects and patients with treated and untreated coeliac disease. Monoclonal antibodies to T lymphocyte surface markers were used to quantify T cell phenotypic subsets in the jejunal mucosa. The patients with untreated coeliac disease had significantly more of both suppressor/cytotoxic and helper/inducer T cells in the surface epithelium than either the control subjects or the treated patients. Serial jejunal biopsy specimens were taken from five treated coeliac patients for six hours after a gluten challenge. In four of these five infiltration of the surface epithelium by both T cell phenotypes, together with deterioration in the villus architecture, had occurred both within two hours of having started the challenge. This suggests that T lymphocytes may have a role in the pathogenesis of coeliac disease.

Detection and estimation of the barley prolamin content of beer and malt to assess their suitability for patients with coeliac disease.

Malt, lager, brown ale, bitter and stout were assessed for their barley prolamin (hordein) content. Monoclonal antibody immunoperoxidase staining of electrophoretically separated samples of beer revealed the presence of immunoreactive hordein. Polyclonal antibody based enzyme linked immunosorbant assays (ELISAs) revealed 1.6 mg of hordein per gram of malt and 1.5 mg per pint in the beers. Patients with coeliac disease should avoid ingestion of beer and foods that contain malt since they contain quantifiable amounts of hordein that is known to exacerbate the condition.

Detection of wheat gliadin contamination of gluten-free foods by a monoclonal antibody dot immunobinding assay.

Unfractionated wheat gliadin was used to produce murine monoclonal antibodies to gliadin. A dot immunobinding assay, using these antibodies, was developed to detect possible gliadin contamination of nominally gluten-free flour, using dilute ethanol extracts spotted onto nitrocellulose membranes. The sensitivity of the assay was less than 10 micrograms/ml of unfractionated gliadin which permitted the detection of trace amounts of gliadin present in certain wheat starch based 'gluten-free' products. The assay detected not only wheat gliadin, but also prolamine extracts of rye, barley and oats; maize, soya and potato extracts as well as the control proteins casein and ovalbumin, gave negative results. The assay is of value as a simple and rapid method of screening foods for their suitability for consumption by patients with coeliac disease.

Q fever encephalitis.

Encephalitis is a rare but documented complication of acute Q fever. We report here the case of a 48-year-old lady who presented with an acute illness characterised by influenza-like symptoms, pneumonia and neurological disturbance but in whom the serology was suggestive of chronic rather than acute Q fever.

Massive hepatosplenomegaly, jaundice and pancytopenia in miliary tuberculosis.

A 29-year-old Caucasian woman presented to hospital with a 2-day history of diarrhoea, anorexia and rigors. Investigations showed abnormal liver function tests, hyponatremia, hypoalbuminaemia and lymphopenia. The initial chest radiograph was normal. A bone marrow trephine biopsy showed non-caseating granulomata and she subsequently developed miliary shadowing on the chest radiograph. A transjugular liver biopsy confirmed the presence of acid-alcohol fast bacilli. Despite starting triple therapy for miliary tuberculosis she remained febrile and developed massive hepatosplenomegaly, jaundice and pancytopenia. Standard triple therapy was substituted with ethambutol, streptomycin and oral prednisolone and the patient made a dramatic recovery. The clinical symptoms of miliary tuberculosis are frequently non-specific and the onset of the illness is often insidious. The liver is involved in almost all patients with miliary tuberculosis, but massive hepatosplenomegaly and jaundice are rare. Standard triple-therapy should be discontinued when there is significant liver dysfunction, and corticosteroids should be considered for patients with miliary tuberculosis who fail to respond to conventional therapy.

Adjuvant itraconazole in the treatment of destructive sphenoid aspergillosis.

Paranasal aspergillosis is a potentially progressive continuum of disease, classically described as having four forms: allergic, non-invasive, invasive, and fulminant. The first two have been considered together as extramucosal disease whilst the latter two are both variants of tissue-invasive disease. Sphenoid aspergillosis, given its anatomical location is a more aggressive disease than that found affecting the other paranasal sinuses, even when non-invasive, and may be fatal. This is compounded by the fact that diagnosis is difficult and so may be made late when aspergillosis is consequently more advanced. Intracranial extension may occur via the direct spread of invasive disease or along communicating veins despite intact sinus walls and lack of fungal mucosal penetration. Once this occurs mortality is high. We have successfully treated three cases of destructive sphenoid aspergillosis, two of which had intracranial extension, with surgery and adjuvant anti-fungal chemotherapy including itraconazole. We recommend the use of post-operative itraconazole in all cases of sphenoid sinus aspergillosis. Additionally, when there is evidence of spread to the brain or other adjacent structures we would advocate an initial course of intravenous amphotericin B followed by long-term oral itraconazole.

Diffuse tuberculous parotitis.

Parenchymatous parotid tuberculosis diffusely affecting the entire gland is very rare. We present a case, associated with a primary pulmonary focus, that was confirmed after positive identification of alcohol and acid-fast bacilli in gastric washings. Both sites of infection resolved with quadruple anti-tuberculous chemotherapy.