The 'Functional Muscle-Bone Unit': probing the relevance of mechanical signals for bone development in children and adolescents.

The present text deals with the relationship of muscle force and mass to bone mass and geometry in the developing skeleton of children and adolescents. Recent results of the last ten years are discussed with reflection on Harold Frost's 'mechanostat hypothesis'. Bone mass and geometry follow the development of body size and muscle force in children and adolescents. Thereby, bone is adapted to the tissue strain due to biomechanical forces. This process is modified by hormonal signals (i.e., estrogens and androgens). Therefore, the quantified relationship of muscle force to bone stability is a reasonable approach to distinguish between primary and secondary bone diseases. Primary bone diseases are characterized by a disturbed adaptation of bone to biomechanical forces. In contrast, secondary bone diseases show a correct adaptation of bone to loaded forces in combination with a decline of muscle force. Therefore, the 'Functional Muscle-Bone Unit' was introduced into the diagnostics of pediatric bone diseases. The ratio of two parameters--referred to bone strength on the one and to biomechanical forces on the other side--is a reasonable diagnostic approach to distinguish between primary and secondary bone diseases.     

Gender difference in cytokine secretion on immune stimulation with LPS and LTA.

Immune defense capacity differs between men and women. Whereas men are more prone to infection and sepsis, women more commonly develop autoimmune diseases. We investigated the difference in cytokine secretion between males and females in response to different immune stimuli. Whole blood from 154 healthy volunteers (age 24 +/- 5.2; 82 females, 72 males) was collected within 2 h on 2 consecutive days. Blood from males produced significantly more tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, and IL-8 than blood from females in response to a high concentration of either lipopolysaccharide (LPS) or lipoteichoic acid (LTA), whereas IL-10 and interferon-gamma (IFN-gamma) secretion did not differ. Normalization of cytokine measurement to individual monocyte counts cancelled these differences for all parameters except TNF-alpha. Stimulation with a lower concentration of LPS (100 pg/mL) produced even stronger differences in cytokine release, which were not cancelled by normalization to the producing cells. The coefficients of variation (CV) of the LPS-induced and LTA-induced cytokine responses were higher in blood from women than men for all parameters and stimuli measured. Thus, the stronger innate immune response of males in comparison to females appears to stem not only from a difference in monocyte counts but also from the steepness of the response curve.     

Laser treatment of eye disorders in children.

The increasing range of indications for laser treatment and the development of new modalities of lasering have led to a growing tendency to extend this type of treatment to the pediatric population. Problems of compliance in this age group often necessitate the use of general anesthesia. This report describes the operative technique used in 11 children (13 eyes) under the age of 13 years, all of whom underwent laser treatment under general anesthesia for a variety of ocular conditions. Argon or krypton laser photocoagulation was performed in eight children (10 eyes), seven of whom were treated for various retinal pathologies and one for an iris cyst. The other three children underwent Nd:YAG posterior capsulotomy for secondary cataract.     

Introduction: lipids and coronary disease--resolved and unresolved problems

To my mind, there are more unresolved problems regarding lipids and cardiovascular diseases than those which have been settled. While this may seem disappointing after 40 years of intensive research, the progress which has been made is remarkable and impressive. It is appropriate, therefore, to begin an introduction by outlining the issues which have been resolved and which are more or less internationally agreed. In doing so, I shall deliberately not digress into causes of coronary heart disease (CHD) other than lipids, even though other influences may be as or more important in relation to its pathogenesis.     

Comparative study between direct mild acid extraction and immunobead purification technique for isolation of HLA class I-associated peptides.

Identification of tumour-specific peptide(s) hidden within the groove of human leucocyte antigens is a crucial prerequisite for peptide vaccine therapy. Conventionally, the peptide(s) are isolated by mild acid extraction (MA) technique followed by sequential ultra-filtrations. Here we describe a new approach for peptide isolation using the immunobead purification (IB-P) technique in conjunction with reverse-phase high-performance liquid chromatography. The obtained data were validated by SDS-PAGE followed by the silver staining technique. The results can be summarised as follows: (1) Comparison of class I-associated peptides isolated from a bladder cell line before and after the correction of class I antigens by gene transfection followed by IB-P technique showed the presence of peptides only from the class I-corrected cells. The data were confirmed using the silver staining technique as a way of detecting individual peptide bands. (2) Whilst peptides could be isolated by both techniques, the MA method led to the isolation of peptides from both class I-negative and class I-positive Fen cell lysates. (3) The IB-P approach could be used for isolation of class I-associated peptides from a normal kidney tissue. The data showed the high efficiency of the IB-P approach for isolation of class I-associated peptides. Unlike the MA technique, where the presence of non- class I-associated peptides was a problem, the IB-P approach isolated only peptides associated with the class I antigens. In addition, the data showed the feasibility of extracting peptides from tissue fragments by the IB-P method. The approach presented here may assist the future development of peptide vaccine therapy in urological cancers.     

DNA transfection of a gene repressing aryl hydrocarbon hydroxylase induction

High mol. wt genomic DNA from a genetically dominant aryl hydrocarbonhydroxylase (AHH)-deficient mutant cell line derived from themouse hepatoma cell line Hepa-1 was used to transfect the parentcell line. AHH-deficient transfectants were recovered followingsingle-step selection in medium containing the carcinogen benzo(a)pyrene.The transfectants arose at a frequency of 2 x 10^–7. Thisfrequency was at least 4-fold greater than the frequency ofspontaneous forward mutation in this cell line. In another setof experiments, dominant mutant DNA was co-transfected alongwith the selectable plasmid pSV2ecogpt into parental Hepa-1cells. The frequency of co-transfection was determined to be3 x 10^–8. This frequency was 150 times greater than thatexpected on the basis of coincident but unrelated spontaneousmutation and plasmid uptake. Both types of transfectants werejudged, following somatic cell hybridizations, to possess thedominant phenotype of the mutant cell line, demonstrating thata trans-acting dominant negative regulator of AHH was transferredin these experiments. DNA transfection should therefore providea means for the molecular cloning of the gene that encodes thedominant regulator.     

Pharmacokinetic and pharmacodynamic studies with 4′-epi-doxorubicin in nasopharyngeal carcinoma patients

Summary The plasma pharmacokinetic profile of 4-epidoxorubicin (epirubicin) was investigated in 28 patients with nasopharyngeal carcinoma (NPC) after single i.v. rapid infusions. All patients had normal liver and renal functions. Plasma concentrations of the parent compound were specifically determined by a high-performance liquid chromatographic (HPLC) method, with UV detection at 254 nm. Plasma levels of the compound were fitted to a three-compartment open model; a triexponential decrease in plasma concentrations with a long terminal plasma halflife (44.8±21.2 h) was observed in 27 patients. The respective mean (±SD) serum concentration at 72 h and the AUC, plasma clearance, and terminal elimination rate constant in complete responders were 7.67±1.98 ng/ml, 4,002±3,080 ng· h/ml, 26.6±12.9 l/h·m², and 0.009±0.007 l/h, whereas those in nonresponders were 4.96±1.8 ng/ml, 1,88±652.8 ng·h/ml, 44.4±15 l/h·m², and 0.017±0.006 l/h, respectively; these differences were significant (P(0.05). Epirubicin produced a 52% response rate, including 6 patients with a complete response, 8 with a partial response, 11 with no change, and 2 with progressive disease. No relationship could be found between the various pharmacokinetic parameters and either leukopenia, age, or sex. These observations strongly suggest that plasma clearance may be one of the determining factors affecting the response or nonresponse of NPC patients to epirubicin, and a dose adjustment according to plasma clearance would probably increase the response rate.     

Impaired microtubule function correctable by cyclic GMP and cholinergic agonists in the Chediak-Higashi syndrome.

The Chediak-Higashi (CH) syndrome of man and several animal species is characterized by the presence of abnormal giant granules in all granule-containing cells and by defects in chemotaxis and lysosomal degranulation during phagocytosis in polymorphonuclear leukocytes (PMNs). Since similar functional abnormalities have been reported in normal PMNs following exposure to colchicine and other agents that disrupt microtubles it was proposed that microtubule function may be impaired in the CH syndrome. The mobility of concanavalin A (con A)-receptor complexes on PMN membranes was used to test microtubule integrity. Normal PMNs showed a uniform distribution of membrane-bound con A. By contrast, con A was aggregated into surface caps on both colchicine-treated normal PMNs and untreated PMNs from mice and a patient with CH syndrome. This result is consistent with impaired microtubule function in the CH cells. The spontaneous capping response of CH PMNs was inhibited by cyclic GMP and by cholinergic agonists that can elevate cyclic GMP levels in neutrophils. This raised the possibility that the microtubule defect in CH cells may be correctable by treatments that increase cyclic GMP generation. Direct evidence for both the absence of microtubule assembly in con A-treated PMNs from the CH patient and for normal microtubule assembly in CH PMNs incubated with cyclic GMP and cholinergic agonists prior to con A treatment was obtained by electron microscopy. In addition, evidence for a direct relationship between the microtubule defect and the development of giant lysosomes in CH cells was obtained. Thus, CH fibroblasts grown in vitro developed abnormal lysosomes in the majority of cells. However, the same cells cultured in the presence of cholinergic agonists developed a majority of lysosomes that were morphologically normal at the level of the light microscope. Similarly, granule morphology appeared normal in peripheral blood leukocytes from mice treated chronically in vivo with cholinergic agonists.     

Identification of a naturally processed cyclin D1 T-helper epitope by a novel combination of HLA class II targeting and differential mass spectrometry

T-helper (Th) cells play an important role in orchestrating the effector function of CTL in anti-tumor immunity. However, only a limited number of Th cell epitopes has been characterized. Here we describe a novel approach for identifying naturally processed and presented peptides derived from chosen antigens. This method combines a transfection step of antigen-presenting cells with a vector encoding a fusion protein between the Ii chain and the antigen of interest, elution of the HLA-bound peptides and identification of the antigen-derived peptides by mass spectrometric comparison to the non-transfected cells. In vitro-stimulated Th cells against the identified peptide of interest specifically recognize transfectants overexpressing the cognate antigen. Using this approach, we were able to identify the HLA-DR4-restricted Th cell epitope NPPSMVAAGSVVAAV derived from cyclin D1, which is frequently overexpressed in tumors. This method will help in identifying peptide candidates for vaccination studies for tumor immunotherapy.