Characterization of T-cell subsets and T-cell receptor subgroups in pigtailed macaques using two- and three-color flow cytometry

In order to characterize macaque T-lymphocyte subsets, we used a chromophore from a dinoflagellate, peridinin chlorophyll A protein (PerCP), which, like fluorescein isothiocyanate (FITC) and R-phycoerythrin (PE), can be excited by a 488-nm laser and emits light at 670 nm without spectral overlap with FITC and PE. Mouse monoclonal antibodies were conjugated with FITC, PE, and PerCP to detect CD4+ and CD8+ cells in macaque peripheral blood lymphocytes (PBL) subsets before and after activation and in nonactivated thymocytes. Resting and activated macaque blood CD4+ T-cells could be clearly delineated into discrete subsets with either CD28, CD45RA, or CD45RO as a second marker and CD26, CD29, CD44, or CD69 as a third marker. CD8+ cells were further subdivided by expression of similar combinations of markers. A subset of CD8+ CD28– T-cells in blood expressed the activation marker CD69, suggesting that they were already activated. Virtually all CD4+CD8+, CD4+CD8–, and CD4–CD8+ macaque thymocytes expressed CD2, CD3, and CD18 and not CD25, CD44, or CD45O, but macaque thymocyte subpopulations did differ in their expression of CD28 and CD29. The expression of T-cell receptor (TCR) subgroups on macaque PBL and thymocytes was analyzed before and after activation with staphylococcal enterotoxins (superantigens). The pattern of T-cell variable-region expression in macaques was similar to that seen in humans, with a high frequency of T cells expressing V8. After superantigen stimulation, only minor changes in TCR V expression were detectable in PBL. A dramatic increase in V8 expression was seen after stimulation of macaque thymus with staphylococcal enterotoxin D (SE-D), a minor increase after toxic shock syndrome toxin 1 (TSST-1) stimulation, and a simultaneous decrease in V6 levels.     

Phenotypic and genotypic characterisation of blood isolates of coagulase-negative staphylococci in the newborn

Coagulase-negative staphylococci (CNS) are the leading cause of late-onset sepsis in newborns (>72 h of age). Our aim was to determine whether phenotypic and/or genotypic differences existed between blood isolates of CNS regarded as inducers of sepsis or as contaminants. Ninety-seven bloodisolates of CNS recovered from newborns at the neonatal intensive care unit, Orebro, Sweden in 1983-1997 were analysed. Twenty-nine of them (30%) were classified as sepsis isolates and 68 (70%) as contaminants. The most prevalent species was Staphylococcus epidermidis (n=59). Staphylococcus haemolyticus (n=16) was most often isolated from newborns with the lowest gestational age and birth weight. Biochemical typing using the Phene Plate system (PhP) and genotyping using pulsed-field gel electrophoresis (PFGE) showed that the S. epidermidis isolates regarded as inducers of sepsis (n=16) were more homogeneous than isolates considered contaminants (n=37). One main genotypic group, representing seven (44%) isolates, was identified among the sepsis isolates. Phenotypically the S. epidermidis sepsis isolates comprised three major clusters. In contrast, among the S. epidermidis contaminants, eight genotypic groups and two phenotypic clusters were identified. The dominating genotypic group among the sepsis isolates of S. epidermidis may represent strains with higher invasive capacity.     

Alterations of mitochondrial function and correlations with personality traits in selected major depressive disorder patients

BACKGROUND: Increased occurrence of several physical conditions has been reported in patients with depressive disorders. Various physical conditions and depressive disorder have been reported in patients with mitochondrial disorders. The aim of this study was to investigate mitochondrial function in selected depressed patients in search of an aetiological or pathophysiological factor common to both depression and physical symptoms. METHODS: Muscle biopsy was performed in 28 patients with a lifetime prevalence of major depressive disorder (MDD), and at least three chronic physical conditions that have been reported to be common in depressive as well as in mitochondrial disorders. Morphologic and biochemical investigations including mitochondrial ATP production rate (MAPR) by the bioluminometric method, spectrophotometric analyses of mitochondrial enzymes, and long-PCR and Southern blot techniques to detect mitochondrial DNA (mtDNA) deletions were performed. The Karolinska Scales of Personality (KSP) with 15 scales assessing vulnerability to psychopathology was filled in by 21 patients. RESULTS: Decreases of MAPR and enzyme ratios were found in the patients in comparison with controls (P<0.01). Deletions of mtDNA assessed with long-PCR were more frequent in patients than in controls (chi-square test P<0.05). Correlations were found between MAPR and the KSP scales 'Somatic Anxiety', 'Psychasthenia', and 'Suspicion' (P<0.01). LIMITATIONS: Results cannot be compared with previous studies, and cannot be generalized to all MDD patients. Individually matched controls were not available. CONCLUSIONS: The results suggest that mitochondrial dysfunction is associated with vulnerability to psychopathology in this selected patient group.     

Die Ausscheidung des ß-Phenylisopropylamins bei Menschen

Zusammenfassung Wir haben bei vier Versuchspersonen die Ausscheidung in den Harn von -Phenylisopropylamin nach peroraler Eingabe untersucht.15–60% der eingegebenen Menge wurde im Harn unverändert wiedergefunden.Die Ausscheidung kann bis 3 Tage nach der Eingabe nachgewiesen werden.Bei größerer Diurese wird verhältnismäßig mehr des eingegebenen Amins unverändert ausgeschieden.Der Prozentteil des ausgeschiedenen Amins scheint von der Größe der eingegebenen Dosis (5–20 mg) unabhängig zu sein.Tägliche Verabreichung von 5 mg während eines Monats zeigt keine Kumulationswirkung.Nach parenteraler Eingabe kann man ß-Phenylisopropylamin im Magen nachweisen.Aus den Versuchen kann mit Sicherheit geschlossen werden, daß jedenfalls ein Teil des ß-Phenylisopropylamins im Menschenorganismus abgebaut oder umgebildet wird, obwohl dies viel langsamer als im Kaninchenorganismus vor sich geht.     

Inter-observer reliability of the “Assessment of Motor Repertoire — 3 to 5 Months” based on video recordings of infants

Objective

A detailed analysis of infant motor behaviour can show up indicators for later neurological impairment. The “Assessment of Motor Repertoire — 3 to 5 Months”, which is part of Prechtl's general movement assessment, could potentially be used for this purpose. The aim of the present study was to investigate inter-observer reliability in this instrument.

Method

Video recordings of 24 infants (corrected ages 3 to 5 months, gestational ages 24 to 42 weeks) were analysed by four observers. Kappa and ICC statistics were applied in the reliability analysis.

Results

High to very high inter-observer reliability was found in the assessment of “Fidgety Movements” (kappa 0.75-0.91). Agreement on the “Movement Character” was also high (kappa 0.54-0.84), while the assessment of the “Posture” showed the lowest inter-observer reliability (kappa 0.39-0.56). Moderate to high inter-observer reliability (kappa 0.51-0.84) was achieved in the field “Quality of Other Movements”, and moderate in “Repertoire of Co-Existent Other Movements” (kappa 0.51-0.69).Inter-observer reliability in the assessment of the total “Motor Optimality Score” was very high between all four observers as intraclass correlation coefficient (2,1) was 0.87, and ICCs for the pairwise analyses ranged between 0.80 and 0.94.

Conclusion

Inter-observer reliability in the “Assessment of Motor Repertoire — 3 to 5 Months” was satisfactory in respect of the subcategories and in case of high and low total optimality scores in pairwise assessments. In the total optimality scores, however, there was some inconsistency in the middle range of the scale.     

Differential effects of UV irradiation on nuclear retinoid receptor levels in cultured keratinocytes and melanocytes

A major risk factor for skin cancer is UV irradiation, which not only damages DNA and other photosensitive compounds like vitamin A, but may also perturb cellular signaling, e.g. via the retinoid receptor system believed to be important for cancer protection. We used cultured normal human keratinocytes and melanocytes to examine the effects of UV irradiation on the expression of the predominant retinoid receptors in the human skin (RARalpha, RARgamma and RXRalpha) and the AP-1 protein c-Jun; mRNA levels were studied by real-time PCR and protein levels by Western blot. In keratinocytes, a single dose of UVB (50 mJ/cm2) caused a rapid drop in the expression of all three receptors (mRNA levels minus 35-50% after 4 h; protein levels minus 20-45% after 8 h), which was followed over the next 40 h by a variable response, leading to full normalization for RARalpha only. In contrast, the levels of c-Jun did not change significantly after UV exposure. In melanocytes, UVB caused a similar drop of the retinoid receptor levels as in keratinocytes but this was soon followed by an increased expression leading to a complete normalization of all receptor levels within 1-3 days. The c-Jun levels in melanocytes increased 1 day after UV exposure and remained high (plus 50%) thereafter. In both cell types, a approximately 3-fold increase in apoptosis (measured by DNA fragmentation) was observed 8-48 h after UVB irradiation. In conclusion, a depletion of vitamin A and retinoid receptors by UV irradiation, together with unchanged or even increased c-Jun levels, might seriously interfere with retinoid signaling and thus promote future tumor development, especially in keratinocytes.