Monellin (MNEI) at 1.15 A resolution.

The X-ray crystal structure of a single-chain monellin protein (MNEI) has been determined at 1.15 A resolution. The model was refined to convergence employing anisotropic displacement parameters and riding H atoms to produce a final model with R(work) and R(free) values of 0.132 and 0.162, respectively. The crystal contains a single MNEI protein in the asymmetric unit and unusually lacks the dimer interface observed in all previous crystal structures of monellin and its single-chain derivatives. The high resolution allowed a more detailed view of MNEI than previously possible, with 38 of the 96 residues modelled with alternative side-chain conformations, including four core residues Thr12, Cys41, Leu62 and Ile75. Four stably bound negative ions were also located, providing new insight into potential electrostatic interactions of MNEI with the largely negatively charged surface of the sweet taste receptor T1R2-T1R3.

Guanine specific binding at a DNA junction formed by d[CG(5-BrU)ACG](2) with a topoisomerase poison in the presence of Co(2+) ions.

The structure of the duplex d[CG(5-BrU)ACG](2) bound to 9-bromophenazine-4-carboxamide has been solved through MAD phasing at 2.0 A resolution. It shows an unexpected and previously unreported intercalation cavity stabilized by the drug and novel binding modes of Co(2+) ions at certain guanine N7 sites. For the intercalation cavity the terminal cytosine is rotated to pair with the guanine of a symmetry-related duplex to create a pseudo-Holliday junction geometry, with two such cavities linked through the minor groove interactions of the N2/N3 guanine sites at an angle of 40 degrees, creating a quadruplex-like structure. The mode of binding of the drug is shown to be disordered, with the major conformations showing the side chain bound to the N7 position of adjacent guanines. The other end of the duplex exhibits a terminal base fraying in the presence of Co(2+) ions linking symmetry-related guanines, causing the helices to intertwine through the minor groove. The stabilization of the structure by the intercalating drug shows that this class of compound may bind to DNA junctions as well as duplex DNA or to strand-nicked DNA ('hemi-intercalated'), as in the cleavable complex. This suggests a structural basis for the dual poisoning of topoisomerase I and II enzymes by this family of drugs.

An analysis of the normal ranges of lymphocyte subpopulations in children aged 5-13 years.

UNLABELLED: Peripheral blood lymphocytes subsets were examined in 233 healthy school children aged 4.9-13.7 years at the time of examination. Lymphocyte subsets were characterized according to the following cluster of differentiation (CD) numbers: CD2, CD4, CD8 and CD19 and quantified according to both their absolute number and as a percentage of total lymphocytes. For the purpose of analysis, the LMS (lambda, mu, sigma) method was utilized, with a 3%-97% confidence interval. Smoothing of the generated curves, was by multiple regression analysis, using the least squares method. The results of these analyses indicate distinct trends as a child ages, both in absolute numbers and in the percentage of each cell type. CONCLUSION: We characterized lymphocyte subsets in children aged 4.8-13.7 years. These data should prove of considerable value to pediatricians dealing with patients with known or suspected immunological problems, and ought to be used in place of the commonly used, but inappropriate, adult lymphocyte subset ranges.

Monitoring cytokine production in peripheral blood during acute graft-versus-host disease following allogeneic bone marrow transplantation.

Plasma concentrations and peripheral blood cells containing cytoplasmic cytokines were monitored during the post-transplant period in 10 patients who had received allogeneic bone marrow transplants (BMT) for the correction of inherited genetic disorders. The presence of CD14-positive cells containing cytoplasmic interleukin-1 alpha and beta in the peripheral blood was indicative of acute graft-versus-host disease (GVHD). Plasma concentrations of IL-1 alpha, IL-1 beta and TNF-alpha were significantly raised in the GVHD group when compared with the uneventful days. There was, however, poor temporal correlation between the plasma concentrations and clinical manifestations of acute GVHD. Cells containing cytoplasmic IL-6 were present in the peripheral blood when patients had clinically suspected and/or microbiologically confirmed infection. The results from this study demonstrate that analysis of peripheral blood cells for cytoplasmic IL-1 alpha and IL-1 beta are better markers of acute GVHD than is monitoring plasma concentrations of these cytokines.

Monitoring of veno-occlusive disease after bone marrow transplantation by serum aminopropeptide of type III procollagen.

Differential diagnosis of hepatic complications after bone marrow transplantation (BMT) is often difficult. To assess whether serum concentrations of the aminopropeptide of type III procollagen (PIIINP) could facilitate diagnosis, we measured serum PIIINP, corrected for age by conversion to standard deviation scores (SDS), serially after BMT in 27 children. A preliminary study of 11 patients showed that a PNIIINP-SDS cutoff of 8.0 was an optimum for diagnosis of veno-occlusive disease (VOD). PIIINP-SDS was increased above cut-off 1-25 days before the onset of clinical signs in the 12 patients (4 from the preliminary group, the others from a group of 16 studied prospectively) who developed VOD, with subsequent changes in PIIINP-SDS mirroring the course of VOD. By contrast, PIIINP-SDS remained below cut-off in all other patients, including 7 with liver graft-versus-host disease and 3 with drug hepatotoxicity. PIIINP-SDS values greater than 8.0 predict, diagnose, and monitor VOD after BMT.

Cytokine gene expression in skin and lymphoid organs in graft versus host disease.

AIM: To determine if human graft versus host disease (GvHD) is associated with any detectable change in cytokine gene expression in the skin and lymphoid organs. METHODS: Reverse transcriptase and the polymerase chain reaction were used to amplify mRNA for interleukins-1 (IL-1), -2 (IL-2), -4 (IL-4) and -6 (IL-6), IL-2 receptor (IL-2R), tumour necrosis factors alpha (TNF-alpha) and beta (TNF-beta), gamma interferon (IFN gamma) and granulocyte macrophage colony stimulating factor (GM-CSF) in frozen punch biopsy specimens of skin and necropsy samples of skin, lymph node, and spleen. RESULTS: No cytokine mRNA was detected in the punch biopsy specimens except weak signals for IL-6 and IL-1 and GM-CSF in two normal donors and IL2-R in one patient with GvHD. In samples of skin taken at necropsy, however, significant quantities of mRNA for TNF-alpha, TNF-beta, and IL-4 were detected in patients who had or had had GvHD in contrast to those without the disease whose skin lacked mRNA for these products but contained detectable quantities of IL-1, IL2-R, IL-6 and GM-CSF. There seemed to be a reciprocal relation between TNF-alpha and IL-4. In necropsy samples of lymph node and spleen a pattern of cytokine production similar to that in the skin was observed with a preponderance of TNF-alpha, TNF-beta and IL-4 in patients with GvHD and GM-CSF and IL-6 in those without the disease. CONCLUSIONS: The local synthesis of these molecules would explain many of the morphological and immunohistological features of GvHD. The failure to detect TNF-alpha, TNF-beta, and IL-4 in skin biopsy specimens exhibiting GvHD is probably due to their small size but further investigations are required.

C-reactive protein and its cytokine mediators in intensive-care patients.

C-reactive protein (CRP) is an acute-phase protein produced by the liver during bacterial infections and inflammation. The cytokines interleukin (IL)-1 beta, IL-6, and tumor necrosis factor (TNF) are widely reported to induce synthesis of CRP by hepatocytes both in vitro and in vivo. We investigated the relation between CRP and its cytokine mediators in 64 critically ill patients during their treatment in the intensive-care unit. Plasma CRP and IL-6 concentrations were significantly lower in patients without any evidence of infection than in those with clinical infection; plasma IL-1 beta concentrations showed no significant difference between any of the groups, but plasma TNF concentrations were lower in patients with evidence of infection. Significant correlation was seen between plasma concentrations of CRP and IL-6 when the latter was measured by bioassay; however, IL-6 showed, at best, only a 50% predictive value for a change in CRP concentration.

Immune changes in peripheral blood resulting from locally directed interleukin-2 therapy in squamous cell carcinoma of the head and neck.

Interleukin-2 (IL-2) was administered locally by constant intra-arterial infusion in four escalating doses from 3 x 10(4)-3 x 10(7) IU/day to 12 patients with squamous cell carcinoma of the head and neck (SCCHN) in a phase I trial. Lymphocyte phenotypic markers and serum cytokine concentrations were measured over the course of treatment. Serum IL-1-alpha, -beta and IL-6 were not induced at any dose level. Tumour necrosis factor (TNF)-alpha was induced in the 2 patients who showed a clinical response (at the lowest dose) as well as in 4/10 of the non-responders. In addition TNF-beta was induced in 3/10 and IFN-gamma in 5/10 non-responders. Soluble IL-2 receptor concentrations were increased at the two higher doses. The highest dose of IL-2 produced a lymphocytosis after day 5 until the end of administration reflected by a general rise in lymphocyte phenotypic markers. CD25, CD3/HLA-DR and CD56 showed an additional upregulation not accounted for by the lymphocytosis with a suggestion of a bell-shaped dose-response curve for CD25 and CD3/HLA-DR. Administration of IL-2 in this manner has been shown to be well tolerated and has some anti-tumour activity at low doses, with little toxicity.

Chronic granulomatous disease 100% corrected by displacement bone marrow transplantation from a volunteer unrelated donor.

A boy whose chronic granulomatous disease (CGD) manifested in infancy, and whose elder brother had died at 7 years of age, had phagocytes with complete lack of functional cytochrome B-245 and which could not be induced by interferon gamma to achieve adequate staphylococcal killing. He underwent an elective displacement bone marrow transplant from a volunteer unrelated donor at the age of 8 months. This has achieved 100% replacement of the CGD granulocytes by those of the normal volunteer and the boy has since had a normal childhood for 3 years. Six previous transplants for CGD are briefly reviewed and illustrate that the host abnormal marrow must be completely displaced using an adequate dose of busulphan to ensure 100% stable engraftment of the donor's marrow and that this is best done under elective conditions before septic foci and irreversible organ damage have occurred. Criteria need to be developed to identify early those patients likely to have severe morbidity.

Post-viral immunodeficiencies in man.

Secondary immunodeficiencies (as defined) of T-cells, B-cells, and phagocytes can be caused by viral infections, and have already been demonstrated for over 20 viruses affecting man. The severity and duration can vary with genetic constitution and age at infection, and post-viral atopy or deregulation permitting triggering of autoimmune diseases are now well-recognised. Various mechanisms are briefly discussed together with some potential treatments, and there is still much to be studied and learned.

Transmission of toxoplasmosis by bone marrow transplant associated with Campath-1G.

A 12-year-old boy in third remission acute lymphoblastic leukaemia was given a mismatched transplant from his mother. He suffered prolonged neutropenia and pyrexia which was only finally diagnosed as toxoplasmosis using molecular biology methods and by his response to appropriate treatment. This was probably transmitted by bone marrow transplant since maternal immune T cells were removed by the use of Campath-1G and treatment with cyclosporin A probably prevented his IgM immune response and impeded the diagnosis.

Bone marrow transplants in genetic diseases.

The first paper [9] advocating the displacement use of bone marrow transplantation (DBMT) to treat a variety of genetic metabolic diseases (including thalassaemia major) was put before a European Working Party in 1978. It evolved from mainly Westminster experience which showed the need [6] for DBMT and first successfully used donors other than matched siblings [9]. The principles of using DBMT to install a donor marrow as a component factory which can last a lifetime are outlined. It is not a panacea, being applicable to only about 7% of known inborn errors. Worthwhile correction of some 50 previously disabling diseases in over 700 patients has already been achieved worldwide and for most of the survivors no further treatment is used after 1 year. Guidelines for future extension, including gene transplants, are offered. The superior results of elective DBMT (about 95%) should encourage paediatricians to aim for earlier diagnoses and evaluations for transplants.

Plasma neopterin as an adjunct to C-reactive protein in assessment of infection.

C-reactive protein (CRP) concentrations are increased in plasma in people with inflammatory conditions and bacterial infections. Plasma neopterin concentrations are increased in people with bacterial septicemias, viral infections, and graft vs host disease. Plasma concentrations of CRP and neopterin were measured daily in 21 bone-marrow transplant (BMT) patients, 64 patients in intensive-care units (ICU), and 12 patients with squamous cell carcinoma of the head and neck (HN). In the BMT patients, plasma neopterin measurements in addition to CRP measurements allowed infectious episodes to be distinguished from graft vs host disease. In the ICU patients, increased concentrations of CRP were not specific for infection and the additional plasma neopterin measurements did not improve this specificity. In all three patient groups, the derivation of a neopterin/CRP ratio was of no clinical use. These three groups of patients showed patterns of CRP and neopterin concentrations characteristic of their underlying diseases, the BMT patients with the immunological activation of graft vs host disease showed predominantly increased concentrations of plasma neopterin, ICU patients with infectious and inflammatory conditions had increased concentrations of both CRP and neopterin in plasma, and the HN group with localized inflammation showed increased plasma concentrations of CRP without increases in neopterin.

A double-blind placebo-controlled study of thymostimulin (TP-1) for the treatment of atopic eczema.

Twenty-nine patients with severe atopic eczema were entered into a randomized double-blind, placebo-controlled trial of the polypeptide thymus extract, thymostimulin (TP-1). The treatment period was 10 weeks with a subsequent follow-up period of up to 1 year. Of the 18 patients receiving TP-1 and the 11 patients on the placebo, 15 and 11 patients, respectively, were fully evaluable. There was no significant difference in either clinical or immunological status between the two treatment groups at baseline. At the 3-month follow-up clinic visit there was a statistically significant difference in the total clinical score, calculated as a percentage of baseline, in favour of the TP-1-treated patients. This difference was not maintained in the subsequent follow-up period and was not accompanied by an improvement in the patient's subjective well-being. There was no significant difference between the treatment groups with respect to the immunological parameters as measured at the 3-month clinic visit.