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LY191145 is a p-chlorobenzyl derivative of LY264826 (A82846B) with activity against both vancomycin-susceptible and -resistant enterococci. Incorporation of L-[14C]lysine into peptidoglycan of intact vancomycin-susceptible and -resistant Enterococcus faecium was inhibited by LY191145 (50% inhibitory concentrations of 1 and 5 microgram/ml, respectively). Inhibition was accompanied by accumulation of UDP-muramyl-peptide precursors in the cytoplasm. This agent inhibited late-stage steps in peptidoglycan biosynthesis in permeabilized E. faecium when either the UDP-muramyl-pentapeptide precursor from vancomycin-susceptible E. faecium or the UDP-muramyl-pentadepsipeptide precursor from vancomycin-resistant E. faecium was used as a substrate. Inhibition of late-stage steps led to accumulation of an N-acetyl-[14C]glucosamine-labeled lipid intermediate indicative of inhibition of the transglycosylation step. Inhibition of peptidoglycan polymerization without affecting cross-linking in a particulate membrane-plus-wall-fragment assay from Aerococcus viridans was consistent with this explanation. The fact that inhibition of peptidoglycan biosynthesis by LY191145 was not readily antagonized by an excess of free acyl-D-alanyl-D-alanine or acyl-D-alanyl-D-lactate ligands indicates that the manner in which this compound inhibits transglycosylation may not be identical to that of vancomycin.

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.

Bacitracin and other antibiotics that inhibit late stages in peptidoglycan biosynthesis induce vancomycin resistance in a high-level, inducibly vancomycin-resistant strain of Enterococcus faecium. Exposure to bacitracin led to synthesis of the lactate-containing UDP-MurNAc-pentadepsipeptide precursor required for vancomycin resistance. These findings indicate that inhibition of peptidoglycan biosynthesis can lead to induction of vancomycin resistance and raise the possibility that multiple signals may serve to induce resistance.

Methods to assess toothbrushing ability vary. The purpose of this study was to determine whether age could be a predictor for toothbrushing ability in children. This study evaluated the brushing patterns of 122 children utilizing the horizontal scrub technique. The results obtained suggest that a child's age is a reasonable predictor for toothbrushing ability.

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.

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.

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 (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.

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.

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.

In the presence of bacitracin, vancomycin-resistant Enterococcus faecium (vanA phenotype) accumulate UDP-N-acetylmuramyl(UDP-Mur-NAc)-tetrapeptide and a UDP-MurNAc-depsipentapeptide containing lactate substituted for the carboxy-terminal-D-alanine residue. In an in vitro peptidoglycan polymerization assay, the modified precursors function and confer resistance to vancomycin.

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.

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.

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.

Daptomycin inhibits the formation of UDP-N-acetylmuramyl-pentapeptide in Bacillus megaterium by inhibiting active transport of amino acids incorporated into the pentapeptide. The ability of daptomycin to inhibit active transport and peptidoglycan formation may be due to its ability to disrupt the transmembrane electrochemical gradient.

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.

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.

A novel staining method for simultaneously determining the immunophenotype and sex of peripheral lymphocytes is described. Cell surface markers on lymphocytes are identified using specific monoclonal antibodies located by a peroxidase anti-peroxidase staining technique (PAP). Lymphocytes are subsequently hybridised with a biotinylated Y chromosome-specific sequence probe and this is located by avidin-biotin-alkaline phosphatase staining. Following the two staining steps the preparation is examined and in the same field lymphocytes show brown peroxidase staining of cell surface markers and red staining of the Y chromosome. The application of this combined staining technique provides a convenient method for studying the development of different cell lineages following sex-mismatched bone-marrow transplantation and for the identification of chimeric situations. The method has been shown to be sensitive and reproducible.

A comparison of paraprotein estimation by scanning densitometry with corresponding immunoglobulin concentration measured by rate immunonephelometry, fixed-time immunonephelometry and kinetic immunoturbidimetry using a large number of sera has indicated overestimation of the immunoglobulin by liquid phase immunoassays in the presence of a monoclonal component. The effect was seen with all three major immunoglobulin isotypes but was most marked with IgM paraproteins when measured by kinetic nephelometry. The overestimations could not be explained by presence of rheumatoid factors or immune complexes in the serum samples, or by non-parallelism, over a range of dilutions, of the reaction of sample and calibrant with the antiserum. The source and species of antiserum used was a major contributory factor.

The pteridine neopterin is a marker of immunological activation and has been shown to be a useful marker of graft-versus-host disease (GVHD) in bone marrow transplant patients. High levels of both neopterin and interferon-gamma (IFN-gamma) were produced in vitro during mixed lymphocyte responses, which may be considered to be a model of the primary events leading to GVHD. Neopterin was shown to be produced by monocytes in response to stimulation with IFN-gamma, but not other cytokines. However, the interleukins IL-1 alpha, IL-1 beta, IL-2, and tumour necrosis factor (TNF) alpha and beta, but not IL-6, stimulated neopterin production by unfractionated peripheral blood mononuclear cells (PBMC), and culture supernatants from PBMC stimulated with IL-1 alpha, IL-1 beta, IL-2 and IL-6, but not TNF-alpha or TNF-beta induced neopterin production following transfer to fresh monocyte cultures. It therefore appears that cytokines may generate neopterin by induction of IFN-gamma, by synergy with low levels of induced IFN-gamma, or by non-IFN-gamma-dependent mechanisms.

Opsonization of Staphylococcus aureus (Oxford strain) and specific IgG subclass antibodies against formalised staphylococci were measured in plasmas from 27 patients with significant S. aureus infections and 35 healthy adults and 15 children. There were no statistically significant differences in the IgG2 and IgG4 levels between two groups and IgG3 was not detected, but the median plasma IgG1 level was significantly higher in patients with staphylococcal infections (P less than 0.00003). The concentration of IgG2 anti-S. aureus antibodies was 25-47 times greater than that of IgG1. If plasmas were decomplemented, the raised IgG1 levels were associated with increased opsonophagocytosis by normal neutrophils (P less than 0.0002).

The initial simple bone marrow transplants for genetic immunodeficiency diseases could hardly be rejected by the host, but required matched sibling donors, only available for about 1 in 5 patients. Improved inductions enabled alternative donors from the family or unrelated volunteers to be used. Measurement of the extent of engraftment by donor cell markers or their normal enzymes showed the need for displacement, which aims to obtain 100% donor-type marrow so that the future immune responses of the recipient become those of the donor and tolerant to donor cells or their products. Immunoprophylaxis can prevent residual host immune cells from surviving to impair the graft. The concept of DBMT with immunoprophylaxis has evolved either to replace abnormal host cells or to confer a component transferable from donor cells to deficient host tissues. Within 10 years over 40 previously fatal genetic diseases have been satisfactorily corrected and seven partially corrected, but for five there has been inadequate delivery of component to genetically defective tissues such as heart, cartilage and brain. The principles can be applied to some 40 other genetic diseases for which no suitable alternative treatments yet exist.

LY173013 and LY186826 are bicyclic pyrazolidinones containing a novel aza-gamma-lactam ring structure. The antibacterial properties of these compounds appear to be related to those of beta-lactam antibiotics in that both classes of compounds share certain common binding molecules such as beta-lactamases and penicillin-binding proteins.

The putative International Federation of Clinical Chemistry immunoglobulin standard, IFCC 74/1, was calibrated against the World Health Organization (WHO) immunoglobulin standard 67/99 by three different methods: automated immunoprecipitation, Laurell rocket immunoelectrophoresis, and radial immunodiffusion. The same antisera were used in all the assays, which were performed in five expert laboratories. With the aid of linearizing transformations for the dilution curves of both materials and use of a carefully weighted statistical evaluation, values in International Units for IgG, IgA, and IgM were ascribed to IFCC 74/1. The values achieved by the three different methods were not statistically different, except in the case of IgA. IgM assays by automated immunoprecipitation were excluded owing to a high degree of imprecision.

The sera from 34 patients with malignant melanoma at various clinical stages of the disease were examined for the presence of circulating immune complexes (CIC) by the C1q solid-phase assay. Their urine and serum samples had been previously examined for the presence of an urinary melanoma-specific protein (MSP) and the corresponding serum antibody. Low levels of CIC (only in the third stage of the disease) and no positive correlation with the presence of MSP were found. The discordance between our and other author's data stresses again the fact that the different laboratory methods for CIC evaluation reveal in a different way the various CIC populations occurring in several diseases.

B lymphocytes from the peripheral blood of 15 melanoma patients and 14 normal adults were studied using immunofluorescence to IgA, IgG, IgD and IgM surface markers. The total number of peripheral blood B lymphocytes was increased in the melanoma patients (1048/mm(3)) compared with normal controls (504) (P<0·01).The expression of excess IgD bearing lymphocytes in these melanoma patients may reflect a derepression of oncofoetal type.

A plasmid conferring resistance to the aminocyclitol antibiotic hygromycin B was isolated from Escherichia coli. The gene conferring resistance to this drug was cloned in pBR322, and the gene was localized to a fragment of ca. 1,510 base pairs. Resistance to hygromycin B is determined by an aminocyclitol phosphotransferase that modifies hygromycin B and structurally related antibiotics. The specific modification of hygromycin B is a phosphorylation of the hydroxyl on the 4 position of the cyclitol ring (hyosamine). The presence of the phosphotransferase in E. coli correlates with reduced accumulation of [14C]hygromycin B.

Clinical preparations of horse antilymphocyteglobulin (ALG) were found to inhibit human bone marrow granulocyte colony growth. This effect was enhanced by complement and was dose dependent, being almost complete at ALG concentrations of 100 microgram/ml. Inhibition was a property of ALG but not of normal horse globulin. However, short incubation of ALG with bone marrow cells occasionally stimulated colony growth and normal horse globulin regularly stimulated it. Three hours' incubation of bone marrow cells with ALG was needed to produce consistent colony inhibition, which was measurable as a reduction in the expected number of colonies and as a fall in the colony: cluster ratio of surviving cell aggregates. Absorption of ALG on acute myeloid leukaemia blast cells removed the inhibiting property of the ALG while preserving its lymphocytotoxic action. Serum from two patients receiving ALG treatment inhibited colony growth for up to 48 hours after ALG administration. The results suggest the presence in ALG of antibodies specifically cytotoxic to myeloid stem cells which may relate to its myleosuppressive properties in vivo, and also indicate that it should be possible to remove antimyeloid antibodies from ALG by absorption. The use of such purified ALG would have advantages in clinical bone marrow transplantation.

Vancomycin resistance in Enterococcus faecium 180, a clinical isolate from England, was studied. Resistance to vancomycin was transferable by conjugation to other enterococci. Expression of resistance was inducible and coincided with the appearance of a new membrane protein.

Serum C-reactive protein concentrations were measured serially during the early transplant period in 68 bone marrow recipients transplanted for leukaemia (34), chronic granulocytic leukaemia (2), severe aplastic anaemia (6), and various inborn errors of metabolism (26). There were 116 clearly documented episodes of infection or acute graft versus host disease or both. Serum C-reactive protein concentrations in patients with viral (11) or fungal infection (6) were normal or only slightly raised. In 32 patients with isolated acute graft versus host disease, only three (10%) showed serum C-reactive protein concentrations above 40 mg/l. Values greater than 40 mg/l were strongly suggestive of bacterial infections and values above 100 mg/l were seen only in patients (43) with bacterial infections with or without acute graft versus host disease. These findings suggest that serum C-reactive protein concentrations are valuable both for diagnosis and monitoring of such infections.

A one-year-old boy with type I H mucopolysaccharidosis (Hurler's disease) was given a bone-marrow transplant (BMT) from his mother in an attempt to replace the deficient enzyme, alpha-L-iduronidase (iduronidase). These is definite evidence of engraftment, the enzyme activity of the recipient's leucocytes reaching heterozygote levels within 37 days of the BMT. Graft-versus-host disease (GVHD) developed but was partially controlled by steroids. From 3-4 months after graft until the present (13 months after the graft) iduronidase activity has been present in the serum and the urine and there has been evidence of considerable degradation of glycosaminoglycans excreted in the urine. The hepatosplenomegaly has disappeared, corneal clouding has cleared, and deterioration in the child's development seems to have been arrested.

High dose methyl prednisolone (HDMP) was used to treat 25 episodes of graft versus host disease (GVHD) in 13 patients after bone marrow transplantation for aplastic anaemia or acute leukemia. All patients showed rapid improvement of GVHD following HDMP with resolutions of gut and skin manifestations over several days, but liver GVHD was less responsive to treatment. The major complication of treatment was infection and four patients died from infection following HDMP. The mode of action of HDMP may be a nonspecific anti-inflammatory effect but it appears to be rapidly effective in controlling GVH reactions.