Appropriate clinical use of human leukocyte antigen typing for coeliac disease: an Australasian perspective.

The past decade has seen human leukocyte antigen (HLA) typing emerge as a remarkably popular test for the diagnostic work-up of coeliac disease with high patient acceptance. Although limited in its positive predictive value for coeliac disease, the strong disease association with specific HLA genes imparts exceptional negative predictive value to HLA typing, enabling a negative result to exclude coeliac disease confidently. In response to mounting evidence that the clinical use and interpretation of HLA typing often deviates from best practice, this article outlines an evidence-based approach to guide clinically appropriate use of HLA typing, and establishes a reporting template for pathology providers to improve communication of results.

Novel Burkholderiales 23S rRNA genes identified in ileal biopsy samples from children: preliminary evidence that a subtype is associated with perianal Crohn's disease.

A novel family of Burkholderiales bacteria was identified in ileal biopsy specimens from children presenting with symptoms of inflammatory bowel disease. A molecular subtyping approach based on sequencing of a variable region of the bacteria's 23S rRNA genes identified three variants. Pilot analysis identified one variant to be significantly associated with perianal Crohn's disease.

Cytotoxicity of human macrophages for tumor cells. Enhancement by human lymphocyte mediators.

Human macrophages, derived from peripheral blood monocytes, acquire enhanced cytotoxicity for human target cells after incubation in mediator-rich supernates from antigen-stimulated lymphocytes. Maximum cytotoxicity was observed after 24-h incubation in mediators. In comparison to normal macrophages, mediator-activated macrophages were cytotoxic to five of the six malignant cell lines tested but had no effect on five nonmalignant cell lines. In 20 experiments with one target (SK-BR-3), mean cytotoxicity was 23 +/- 2.7% and with another target (MA-160), was 29 +/- 3.4%. Macrophages became cytotoxic after 8-h incubation with mediators and the enhanced cytotoxicity persisted for at least 40 h after the lymphocyte mediators were removed. These findings are consistent with the hypothesis that macrophages, activated by antigen-induced lymphocyte mediators, can contribute to the host resistance to tumor growth in man.

Biochemical purification of antineoplastic agents obtained from sturgeon serum.

Discontinuous and continuous DEAE-Sephadex ion-exchange gradients were utilized to purify two sturgeon serum factors (A2 and B2) that possessed cytotoxic activity in vitro against human tumor cells but not against normal cells. After rabbit antisturgeon factor A2 or B2 was prepared, the antibodies were insolubilized. Sturgeon serum then was adsorbed to the insolubilized antibodies, and factors A2 and B2 were isolated in quantities large enough to conduct biologic studies. When the two factors purified either by ion-exchange chromatography or immunoadsorbent columns were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, factor A2 consisted of one protein band with a molecular weight greater than 68,000 and an isoelectric point of approximately 6.5. Likewise, factor B2 also consisted of one protein band with a molecular weight in excess of 68,000 and an isoelectric point of approximately 7.5. Both factors A2 and B2 were destroyed either by heating at 56 degrees C for 30 minutes or by heating at 100 degrees C for 2 minutes. Furthermore, the mechanism of tumor killing by the sturgeon factors appears to be by means of a soluble factor. When the kinetics of tumor killing was studied, the sturgeon factors were noncytotoxic after 4 or 8 hours of incubation and became cytotoxic after 24 hours of incubation.

A prospective study of delirium and prolonged hospital stay. Exploratory study.

Using explicit criteria, delirium was diagnosed in 15% of a cohort of 133 hospitalized patients. Following each patient's discharge or death, the length of stay was compared with the diagnosis related group-predicted length of hospitalization. An analysis of stay variations disclosed that delirious patients exceeded their predicted stay by an average of 13 days, while nondelirious patients exceeded theirs by 3.3 days. The mean (+/- SD) length of hospitalization for patients with delirium was significantly longer than for their nondelirious counterparts (21.6 +/- 23.7 days vs 10.6 +/- 10.1 days, respectively). Hospitals treating high proportions of patients with delirium as a comorbidity to a principal somatic diagnosis should institute measures for the early detection of and appropriate intervention in patients with this condition. These steps may help reduce prolonged hospitalizations and minimize financial risk under the current diagnosis related group reimbursement system.

In vivo macrophage-mediated tumor cytotoxicity in the mouse.

Tumor-bearing animals injected with either LPS-activated or control macrophages were capable of suppressing tumor growth in vivo. Tumor growth rate and survival times were assessed for each group of animals. At day 12 after injection of the P815 tumor cells, no difference in tumor size could be demonstrated in any of the groups. However, by day 17 the tumors in the animals treated with 2.5 X 10(6) or 5 X 10(6) control as well as LPS-activated macrophages did not continue to increase in size as was seen in the case of the control animals. When examining survival times, it appeared that the animals treated with control or activated macrophages generally survived approximately 10 days longer than did the animals receiving no treatment (30-day vs 20 day survival). Thus, it appears that tumor cell growth can be slowed down in vivo when control or activated macrophages are injected into the site of the tumor mass.

Specificity of macrophage-mediated cytotoxicity: role of target and effector cell fucose.

In this study, we have examined the role cell surface carbohydrates play in the activation of macrophages to kill tumor cells as well as the effect removal of tumor cell surface fucose has on the susceptibility of the treated target cells to the cytotoxic macrophages. After incubation of human monocyte-derived macrophages with alpha-L-fucosidase, a glycosidase which splits terminal alpha-L-fucose from oligosaccharides, the macrophages were no longer able to respond to lipopolysaccharide (LPS). These experiments strongly suggest that alpha-L-fucose probably comprises an essential part of the macrophage membrane receptor for LPS. In another series of experiments tumor cells were treated with alpha-L-fucosidase prior to co-cultivation with macrophages to determine whether the presence of alpha-L-fucose on the tumor cell surface had any effect on macrophage mediated cytotoxicity. It was found that the MA-160 sensitive tumor target, after alpha-L-fucosidase treatment, became resistant to the effects of the cyto-toxic macrophages whereas the effect of the alpha-L-fucosidase treatment was blocked by addition of alpha-L-fucose to the incubation mixture.

Androgen and glucocorticoid regulation of androgen receptor cDNA expression.

Androgen receptor (AR) levels are regulated by androgens, other steroids and non-steroidal hormones via complex, tissue-specific processes. Since alterations in receptor levels may influence cellular sensitivity to androgens, understanding AR regulation is of fundamental and potentially therapeutic significance. In most target tissues and AR-containing cell lines, AR mRNA is down-regulated in response to androgens. We have reconstituted this androgen-mediated down-regulation of AR mRNA in COS 1 cells transfected with a human AR cDNA under the control of the cytomegalovirus (CMV) promoter. The sequences mediating receptor mRNA down-regulation are represented within the AR cDNA and not within the CMV promoter. Androgenic down-regulation of AR cDNA expression was time- and dose-dependent, resembling native AR mRNA down-regulation. In addition, androgenic regulation of the receptor cDNA was not dependent on protein synthesis suggesting that AR and/or another pre-existing protein(s) is involved in this process. In COS 1 cells co-transfected with androgen and glucocorticoid receptor cDNAs, dexamethasone mimicked the action of androgen in down-regulating AR mRNA. This response depended on glucocorticoid receptors. Androgen had little effect on steady-state levels of AR protein consistent with reports that androgen down-regulates AR mRNA but increases AR protein half-life (Kemppainen et al. (1992) J. Biol. Chem. 267, 968-974; Zhou et al. (1995) Mol. Endocrinol. 9, 208-218). However, glucocorticoids decreased AR protein levels in cells that co-expressed androgen and glucocorticoid receptors. These results indicate that sequences represented in the AR cDNA mediate AR mRNA down-regulation by both androgens and glucocorticoids. Inhibition of AR mRNA and protein by glucocorticoids suggests that these steroids may modulate androgen action in tissues, such as mammary gland and prostate, which express both androgen and glucocorticoid receptors.

Delirium: a test of the Diagnostic and Statistical Manual III criteria on medical inpatients.

Although 10% to 15% of patients admitted to acute care hospitals are in a state of delirium, few patients are given this diagnosis by their clinician. We field-tested the Diagnostic and Statistical Manual III (DSM-III) criteria for diagnosing delirium on 133 consecutively admitted patients to an acute medical ward. Twenty patients were delirious using DSM-III criteria, 19 more patients than were reported by the primary clinician. Seven delirious patients were less than 65 years of age (range, 32 to 64 years). Sixty-five percent of patients with delirium died, whereas significantly fewer (3.3%) of patients without delirium died (P less than .0001). We found that delirium could be readily and reliably detected (kappa coefficient of agreement = 0.62 for interrater reliability) using the DSM-III criteria. Clinicians should routinely screen hospitalized patients of all ages using DSM-III criteria to identify delirious patients for an immediate evaluation and treatment.

Macrophage and polymorphonuclear leukocyte function in patients with Alzheimer disease.

Monocyte derived macrophages and polymorphonuclear leukocytes (PMNs) isolated from the peripheral blood of thirteen patients with Alzheimer disease were studied for their cytotoxic effects on a sensitive allogenic tumor target. PMN cells from 11 of the 13 patients with Alzheimer disease were able to kill the tumor cells. In addition, the macrophages from 12 of the 13 Alzheimer disease patients were cytotoxic towards the tumor targets. Four of these patients possessed a plasma inhibitory factor which was capable of suppressing macrophage mediated cytotoxicity. When the lymphocytes from these patients were studied for their ability to be stimulated with the specific antigen, streptokinase, to produce macrophage activating factor (MAF), only 5 of the 13 patients studied possessed lymphocytes which were capable of producing MAF. Thus, the only immunological defect in Alzheimer disease patients which was observed in this study was in the ability of the lymphocytes to synthesize MAF.

Agreement of aspiration tests using barium videofluoroscopy, salivagram, and milk scan in children with cerebral palsy.

To study the agreement between three tests for aspiration, barium videofluoroscopy, salivagram, and milk scan we studied 63 children with severe non-ambulant spastic quadriplegic cerebral palsy (CP) aged 14 months to 16 years (32 males, 31 females). The salivagram was most frequently positive (56%, 95% confidence interval 43 to 68%); the next most frequently positive was barium videofluoroscopy when aspiration was defined as the presence of either laryngeal penetration of material or frank aspiration (39%, 95% confidence interval 26 to 53%). The milk scan was rarely positive (6%, 95% confidence interval 2 to 16%). Agreement between the tests of aspiration was poor. The maximum agreement (kappa=0.20) was between aspiration as diagnosed with the salivagram and by barium videofluoroscopy. Positive tests for aspiration are frequent in children with severe CP. Frequency varies widely depending on the investigation used. There is poor agreement between tests used for the diagnosis of aspiration. This information is of importance in assessing the significance of test results.

Lipofuscin accumulation, abnormal electrophysiology, and photoreceptor degeneration in mutant ELOVL4 transgenic mice: a model for macular degeneration.

Macular degeneration is a heterogeneous group of disorders characterized by photoreceptor degeneration and atrophy of the retinal pigment epithelium (RPE) in the central retina. An autosomal dominant form of Stargardt macular degeneration (STGD) is caused by mutations in ELOVL4, which is predicted to encode an enzyme involved in the elongation of long-chain fatty acids. We generated transgenic mice expressing a mutant form of human ELOVL4 that causes STGD. In these mice, we show that accumulation by the RPE of undigested phagosomes and lipofuscin, including the fluorophore, 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetraenyl]-1-(2-hyydroxyethyl)-4-[4-methyl-6-(2,6,6,-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]-pyridinium (A2E) is followed by RPE atrophy. Subsequently, photoreceptor degeneration occurs in the central retina in a pattern closely resembling that of human STGD and age-related macular degeneration. The ELOVL4 transgenic mice thus provide a good model for both STGD and dry age-related macular degeneration, and represent a valuable tool for studies on therapeutic intervention in these forms of blindness.

Upper and lower gastrointestinal endoscopy in children and adolescents with Crohn's disease: a prospective study.

Fifty-six children and adolescents with Crohn's disease were prospectively investigated with gastroscopy and colonoscopy irrespective of localizing symptoms or signs. Routine biopsies were taken from endoscopically normal and abnormal areas. A high incidence (71%) of upper gastrointestinal (GI) involvement was found. In 41%, these findings were instrumental in making the diagnosis. The ileum was viewed in 49 of the 56 cases. Overall, the upper GI tract was involved in 71%, the terminal ileum in 53%, and the colon in 86% (oesophagus 16%, body of stomach 46%, antrum 36%, duodenum 21%, terminal ileum 53%, caecum 69%, transverse colon 71%, sigmoid 60% and rectum 41%). Upper and lower gastrointestinal endoscopy with systematic biopsies should be performed early in the diagnostic assessment of children and adolescents with suspected inflammatory bowel disease to enable accurate diagnosis and assessment of extent of disease.

Effect of neuropeptides on macrophage mediated cytotoxicity in normal donors and cancer patients.

The role of enkephalins, endorphins and other neuropeptides produced by the nervous system in the alteration of immune responsiveness is generally unknown. The present studies were undertaken to investigate the role of these neuropeptides in the modulation of cytotoxicity induced by LPS activated macrophages obtained from normal donors as well as breast cancer and Hodgkins disease patients. When the macrophages from normal donors were pretreated with these neuropeptides for 1 hr prior to co-culturing with target cells, macrophage mediated cytotoxicity was enhanced with 10(-6) M and 10(-8) M of [met]-enkephalin, 10(-6) M of [leu]-enkephalin and 10(-6) M and 10(-12) M of alpha-endorphin. However, when the macrophages were co-cultured with target cells in the presence of the neuropeptides, it was observed that 10(-6) M and 10(-12) M of alpha-endorphin enhanced cytotoxicity whereas no enhancement in cytotoxicity was observed when [met]-enkephalin or [leu]-enkephalin were added to the cultures. In fact, it appears that 10(-10) M of [met]-enkephalin and 10(-12) M of [leu]-enkephalin actually suppressed macrophage mediated cytotoxicity. When the opioid antagonist Naloxone was incubated with the neuropeptides in the presence of the macrophages the enhancement of macrophage killing produced by [met]-enkephalin, [leu]-enkephalin or alpha-endorphin was suppressed. When the breast cancer patients' macrophages were pretreated with these neuropeptides, enhancement in cytotoxicity was observed at 10(-10) M of [met]-enkephalin, and [leu]-enkephalin and at 10(-8) M and 10(-12) M of alpha-endorphin.(ABSTRACT TRUNCATED AT 250 WORDS)

Lidocaine inhibits macrophage mediated cytotoxicity and enhances neutrophil mediated cytotoxicity.

Peripheral blood monocyte derived macrophages and polymorphonuclear leukocytes (PMNs) obtained from normal donors kill tumor cells in vitro. However, if lidocaine is added to the macrophage tumor cell suspensions in microgram concentrations (0.1 micrograms-0.1 mg), there is marked inhibition of tumor cell killing. The inhibitory effect for the macrophages resulted from an effect of lidocaine on the effector cells. When the effector cells were preincubated with lidocaine, inhibition in macrophage mediated cytotoxicity was observed. Cytotoxicity was also inhibited when 0.01 mg of lidocaine was added to the macrophage monolayers either at the time of addition of the tumor cells or 15-60 min after addition of the tumor cells whereas no inhibition of cytotoxicity occurred when lidocaine was added more than 60 min after the initiation of the cytotoxic reaction. For the neutrophils it was observed that the lidocaine actually had an enhancing effect rather than inhibitory effect on their ability to kill tumor cells.

Role of hydrocortisone, chloroquine and prednisolone in neutrophil mediated cytotoxicity.

Human polymorphonuclear leukocytes (PMNs) obtained from normal donors kill tumor cells in vitro. This system was utilized to study the role of lysosomal enzyme release in the cytotoxicity of human neutrophils. When the neutrophils were incubated with hydrocortisone, chloroquine and prednisolone, which are drugs known to stabilize lysosomal membranes, hydrocortisone and chloroquine were unable to inhibit cytotoxicity whereas treatment with prednisolone inhibited cytotoxicity. Recovery in cytotoxicity was observed as the dose of prednisolone utilized was decreased. Cytotoxicity equivalent to that observed with no added drug was found 5 X 10(-7) M for prednisolone. Pretreatment of neutrophils with a dose of prednisolone known to inhibit cytotoxicity (5 X 10(-6) M) resulted in decreased release of the lysosomal enzyme, acid phosphatase, in comparison to untreated neutrophils.