Sulfide-inhibition of mitochondrial respiration at very low oxygen concentrations.

Our aim was to study the ability of an immortalized cell line (AMJ2-C11) to sustain aerobic cell respiration at decreasing oxygen concentrations under continuous sulfide exposure. We assumed that the rate of elimination of sulfide through the pathway linked to the mitochondrial respiratory chain and therefore operating under aerobic conditions, should decrease with limiting oxygen concentrations. Thus, sulfide's inhibition of cellular respiration would occur faster under continuous sulfide exposure when the oxygen concentration is in the very low range. The experiments were performed with an O2K-oxygraph (Oroboros Instruments) by suspending 0.5-1×10(6) cells in 2 ml of continuously stirred respiration medium at 37 °C and calculating the oxygen flux (JO2) as the negative derivative of the oxygen concentration in the medium. The cells were studied in two different metabolic states, namely under normal physiologic respiration (1) and after uncoupling of mitochondrial respiration (2). Oxygen concentration was controlled by means of a titration-injection pump, resulting in average concentration values of 0.73±0.05 µM, 3.1±0.2 µM, and 6.2±0.2 µM. Simultaneously we injected a 2 mM Na2S solution at a continuous rate of 10 µl/s in order to quantify the titration-time required to reduce the JO2 to 50% of the initial respiratory activity. Under the lowest oxygen concentration this effect was achieved after 3.5 [0.3;3.5] and 11.7 [6.2;21.2]min in the uncoupled and coupled state, respectively. This time was statistically significantly shorter when compared to the intermediate and the highest O2 concentrations tested, which yielded values of 24.6 [15.5;28.1]min (coupled) and 35.9 [27.4;59.2]min (uncoupled), as well as 42.4 [27.5;42.4]min (coupled) and 51.5 [46.4;51.7]min (uncoupled). All data are medians [25%, and 75% percentiles]. Our results confirm that the onset of inhibition of cell respiration by sulfide occurs earlier under a continuous exposure when approaching the anoxic condition. This property may contribute to the physiological role of sulfide as an oxygen sensor.

[Frequency of hearing disorders in children with langerhans' cell histiocytosis]. / Häufigkeit von Hörstörungen bei Langerhans-Zell-Histiozytose im Kindes- und Jugendalter.

BACKGROUND: The aims of the study were to find out the frequency of hearing disorders in children with Langerhans' cell histiocytosis (LCH) and to find out possible risk factors for hearing disorders due to the disease itself and the therapy. PATIENTS AND METHODS: 30 patients with LCH were examined audiologically by using standard audiometric procedures. In cases of central nervous system (CNS) lesions, brainstem evoked response audiometry (BERA) was done additionally. A significant hearing disorder was defined as a hearing impairment affecting speech, thus being characterized by the following features: Both ears should present either conductive and/or sensorineural hearing loss of at least moderate degree. The frequencies important for speech development (1 - 4 kHz) had to be affected. Also, prolongation of BERA interpeak latencies was regarded as a significant hearing impairment, because those children had an increased risk for a central auditory and speech processing disorder. RESULTS: In our study 3 patients had a significant hearing disorder according to our definition. The prevalence of a significant hearing disorder is increased in patients with LCH compared to the prevalence of permanent hearing disorders in German children. We found the following risk factors for the development of a significant hearing disorder: LCH of the temporal bone, CNS lesions. CONCLUSIONS: We recommend periodical audiological follow up examinations for LCH patients with lesions of the temporal bone and/or the brain. BERA is indicated in cases with CNS lesions, because central auditory and speech processing disorders are possible in those patients.

Diphtheria (D) and tetanus (T) antibody values in children with acute lymphoblastic leukaemia (ALL) after treatment according to Co-ALL 05/92.

BACKGROUND: Children and adolescents after acute lymphoblastic leukemia are at risk for a prolonged period of immunodeficiency. Normally within 6 to 9 months after the end of maintenance treatment an adequate immune recovery is present. Factors such as immunity against specific antigens prior to disease (applied baseline vaccination), intensity of treatment and age can play a role in the appearance of antibodies in serum. Diphtheria (D) and Tetanus (T) antibodies are known to appear within 3 to 6 months after end of treatment as a sign of immune recovery and the reinstatement of immunological memory. A number of different questions are of interest: What differences are seen in the antibodies to D and T in children of different ages after treatment with a standardized protocol? What is the influence of post-treatment revaccination with Diphtheria/Tetanus (D/T) and treatment group on the production of D/T antibodies? PATIENTS AND METHODS: Out of 142 children and adolescents until the age of 16, treated according to the Co-ALL 05/92 protocol, 59 patients were eligible for evaluation: 31 Low-Risk (LR)- and 28 High-Risk (HR) patients. Antibodies against Diphtheria (D) and Tetanus (T) were measured 3-12 months after the end of treatment and after revaccination in case of low antibody levels against D and/or T. In patients without adequate response after repeated revaccination the cellular immunity was examined with a skin test. RESULTS: After the end of treatment, children in the low-risk (LR)-group showed more frequently adequate antibody titres against D and T than children of the high-risk (HR)-group. Antibodies against T were present in 50% of all patients. After revaccination antibodies against T were found in nearly all patients whereas for D this is only the case in some children. Patients without sufficient antibody levels mainly showed an adequate cellular immunity. CONCLUSION: In children and adolescents with ALL after therapy antibody levels of D and T are dependent on treatment intensity. Revaccination leads to an adequate immunological answer against T in most patients , which is not the case for the diphtheria vaccination. Prospective multicenter trials starting together with the ALL-treatment should be able to gain more information about the behavior of antibody levels and the risk of infection from vaccine-preventable disease in immunocompromised patients and thus lead to standardized vaccination guidelines such as immunization with conjugate vaccines already during maintenance treatment.

Supravenous hyperpigmentation, transverse leuconychia and transverse melanonychia after chemotherapy for Hodgkin's disease.

Pigmentary abberations of the skin, mucosa and epidermal appendages are common side-effects after systemic treatment with chemotherapeutic agents. These pigment changes appear in different patterns and are partly quite typical for the applied chemotherapeutic drug. The pathogenesis of the different skin pigmentations are not well known. The most often discussed causes are the stimulation of melanocytes, involvement of the tyrosinase enzyme system and thrombophlebitis with postinflammatory hyperpigmentation by the aggressive substances. Nail discolorations are mainly due to direct toxic effects and stimulation of the matrix melanocytes. We report a rare event of supravenous hyperpigmentation, transverse leuconychia and melanonychia after chemotherapy of a patient suffering from Hodgkin's disease.

Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized cell lineages.

A plexus of lymphatic vessels guides interstitial fluid, passenger leukocytes, and tumor cells toward regional lymph nodes. Microvascular endothelial cells (ECs) of lymph channels (LECs) are difficult to distinguish from those of blood vessels (BECs) because both express a similar set of markers, such as CD31, CD34, podocalyxin, von Willebrand factor (vWF), etc. Analysis of the specific properties of LECs was hampered so far by lack of tools to isolate LECs. Recently, the 38-kD mucoprotein podoplanin was found to be expressed by microvascular LECs but not BECs in vivo. Here we isolated for the first time podoplanin(+) LECs and podoplanin(-) BECs from dermal cell suspensions by multicolor flow cytometry. Both EC types were propagated and stably expressed VE-cadherin, CD31, and vWF. Molecules selectively displayed by LECs in vivo, i.e., podoplanin, the hyaluronate receptor LYVE-1, and the vascular endothelial cell growth factor (VEGF)-C receptor, fms-like tyrosine kinase 4 (Flt-4)/VEGFR-3, were strongly expressed by expanded LECs, but not BECs. Conversely, BECs but not LECs expressed VEGF-C. LECs as well as BECs formed junctional contacts with similar molecular composition and ultrastructural features. Nevertheless, the two EC types assembled in vitro in vascular tubes in a strictly homotypic fashion. This EC specialization extends to the secretion of biologically relevant chemotactic factors: LECs, but not BECs, constitutively secrete the CC chemokine receptor (CCR)7 ligand secondary lymphoid tissue chemokine (SLC)/CCL21 at their basal side, while both subsets, upon activation, release macrophage inflammatory protein (MIP)-3alpha/CCL20 apically. These results demonstrate that LECs and BECs constitute stable and specialized EC lineages equipped with the potential to navigate leukocytes and, perhaps also, tumor cells into and out of the tissues.

Cloning and expression of secretagogin, a novel neuroendocrine- and pancreatic islet of Langerhans-specific Ca2+-binding protein.

We have cloned a novel pancreatic beta cell and neuroendocrine cell-specific calcium-binding protein termed secretagogin. The cDNA obtained by immunoscreening a human pancreatic cDNA library using the recently described murine monoclonal antibody D24 contains an open reading frame of 828 base pairs. This codes for a cytoplasmic protein with six putative EF finger hand calcium-binding motifs. The gene could be localized to chromosome 6 by alignment with GenBank genomic sequence data. Northern blot analysis demonstrated abundant expression of this protein in the pancreas and to a lesser extent in the thyroid, adrenal medulla, and cortex. In addition it was expressed in scant quantity in the gastrointestinal tract (stomach, small intestine, and colon). Thyroid tissue expression of secretagogin was restricted to C-cells. Using a sandwich capture enzyme-linked immunosorbent assay with a detection limit of 6.5 pg/ml, considerable amounts of constitutively secreted protein could be measured in tissue culture supernatants of stably transfected RIN-5F and dog insulinoma (INS-H1) cell clones; however, in stably transfected Jurkat cells, the protein was only secreted upon CD3 stimulation. Functional analysis of transfected cell lines expressing secretagogin revealed an influence on calcium flux and cell proliferation. In RIN-5F cells, the antiproliferative effect is possibly due to secretagogin-triggered down-regulation of substance P transcription.