Kindler syndrome. Clinical and ultrastructural findings

BACKGROUND: Kindler syndrome is a genodermatosis that combines clinical features of hereditary epidermolysis bullosa and poikiloderma congenitale. The ultrastructural level of blister formation has not been well characterized. OBSERVATIONS: Two brothers with Kindler syndrome had a history of primarily acral blistering since infancy as well as photosensitivity. Blister formation was found through the basal layer. Marked tonofilament clumping was found in intact keratinocytes adjacent to the blisters. The younger brother (aged 21 years) had actinic keratoses, which have not been previously described in Kindler syndrome. CONCLUSIONS: The findings of basal layer separation in both spontaneous and induced blisters in Kindler syndrome suggest this is the true level of blister formation. The finding of actinic keratoses in a young patient with Kindler syndrome suggests that some patients may be at increased risk for early solar-induced skin disease. The presence of clumped tonofilaments in keratinocytes adjacent to blistered areas suggests an abnormality of keratin 5 or 14 could be present and may play a role in blister formation in patients with Kindler syndrome.     

Capillary electrophoresis combined with matrix-assisted laser desorption/ionization mass spectrometry; continuous sample deposition on a matrix-precoated membrane target

Capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) were combined in an off-line arrangement to provide separation and mass analysis of peptide and protein mixtures in the attomole range. A membrane target, precoated with MALDI matrix, was used for the continuous deposition of effluent exiting from a CE device. A sample track was produced by linear movement of the target during the electrophoretic separation and this track was subsequently analyzed by MALDI/MS. The technique is effective for peptides and proteins, having limits of detection (signal-to-noise >3) of about 50 amol for neurotensin (1673 Da) and 250 amol for cytochrome c (12361 Da) and apomyoglobin (16951 Da). The electrophoretic separation achieved from the membrane target, as measured by theoretical plate numbers from the mass spectrometric data, can be as high as 80-90% of that achieved by on-line UV detection under optimal conditions, although band broadening occurs and with some loss of separation efficiency. Non-volatile buffers such as 10-50 mM phosphate can also be used in the electrophoresis process and directly deposited on the membrane. The use of post-source decay techniques is shown for peptides in the CE sample track in order to obtain sequence verification. The effectiveness of this method of integration of CE and MALDI/MS is demonstrated with both peptide and protein mixtures and with the analysis of a tryptic digest of a protein.     

Performance of a self-administered mailed version of the Quality of Well-Being (QWB-SA) questionnaire among older adults

OBJECTIVES: The Quality of Well-Being questionnaire is a measure of health-related quality of life (HRQoL) that has several desirable properties. Its widespread use has been hindered because it is difficult to administer. To overcome this limitation, a new self-administered form has recently been developed. This study examined the feasibility of using the Quality of Well-Being-Self-Administered (QWB-SA) questionnaire in an older population. METHODS: The Quality of Well-Being-Self-Administered questionnaire was sent to 430 community-dwelling individuals aged 65 years and older who were randomly selected from primary care physicians' offices. Response patterns, scaling distributions, and the acceptability of the survey were examined for all respondents. The results of the QWB-SA questionnaire were compared to the Sickness Impact Profile (SIP) and the Medical Outcomes Study 36-item Short-Form Health Survey (SF-36) for those individuals who also had completed the latter two surveys approximately 10 months earlier and whose health had not changed substantially in the meantime. RESULTS: Three hundred and one older adults (70%) responded. The mean QWB-SA questionnaire score was 0.7035. The scores were not skewed, and there were no floor or ceiling effects. The mean time to complete the QWB-SA questionnaire was 14.2 minutes, which was significantly shorter than for the SIP (19.3 minutes) but significantly longer than for the SF-36 (12.5 minutes). Subjects rated their satisfaction with the QWB-SA questionnaire somewhat lower than for the SIP and similar to SF-36. Correlations between the QWB-SA questionnaire and the SIP and SF-36 were moderate and were generally stronger for measures of physical health than for other domains such as mental health. CONCLUSIONS: The self-administered QWB questionnaire was acceptable to older respondents, and it correlated with other measures of health-related quality of life. It can be considered as a candidate for some research applications among older adults.     

Nimodipine monotherapy and carbamazepine augmentation in patients with refractory recurrent affective illness

We investigated the expression and function of Fas and Fas ligand (FasL) on peripheral blood lymphocytes (PBLs). The cells were stimulated with various cytokines or 12-0-tetradecanoyl phorbol 13-acetate (PMA) plus ionomycin. About 30% of unstimulated PBLs expressed Fas, and the expression was augmented by interleukin-1beta (IL-1beta), IL-2, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), or PMA plus ionomycin. Although only minimal FasL expression was detected on unstimulated PBLs, FasL expression was markedly induced by IL-2 or PMA plus ionomycin, suggesting that Fas and FasL were both expressed on IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs. Although IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs were positive for both Fas and FasL, no significant increase in apoptosis was demonstrated in these activated PBLs. In addition, treatment of PBLs with IL-2 or PMA plus ionomycin did not change anti-Fas-induced apoptosis, although these activated PBLs expressed Fas strongly when compared with unstimulated PBLs. Only IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs killed Fas+ target cells efficiently via the interaction of Fas on target cells with FasL of PBLs. Bcl-2 was constitutively expressed on unstimulated PBLs, but its expression was significantly augmented by IL-2 or PMA plus ionomycin. The expression of Bax was clearly induced only on IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs and that of other Bcl-2 family proteins such as Bcl-x and Bad could not be detected on human PBLs, including IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs. Our results suggest that PBLs activated by IL-2 or PMA plus ionomycin express both Fas and FasL and that they kill Fas+ target cells by using FasL on the surface. The resistance of these activated PBLs to Fas-mediated apoptosis may be due to the augmented Bcl-2 expression or the presence of Bcl-2:Bax heterodimers on these cells.     

Transgenic mice for the establishment of histidinol-resistant embryonic fibroblast feeder layers

Gene targeting in mouse embryonic stem cells generates mutations by replacing an endogenous chromosomal region with a copy disrupted by a selectable genetic marker. The most commonly used selectable marker is the bacterial neo(r) gene, which confers resistance in mammalian cells to the antibiotic G418. Use of an alternative selectable marker, the Salmonella typhimurium gene hisD, should provide expanded applications for gene targeting. The hisD gene encodes the protein histidinol dehydrogenase, which catalyzes the conversion of histidinol to the amino acid histidine. Histidinol is toxic to mammalian cells, while histidine is an essential mammalian amino acid. Consequently, growth selection in cultures with media containing histidinol in place of histidine occurs by both histidine starvation and histidinol poisoning. The hisD selection is being tested for potential use in gene targeting experiments with mouse embryonic stem (ES) cells. Currently, most successful gene targeting experiments use primary embryonic fibroblast feeder layers, which assist in the maintenance of the pluripotential state of the embryonic stem cells. To support ES cell stability under histidinol selection, mice transgenic for the S. typhimurium hisD gene have been produced and used to generate embryonic fibroblast feeder cells. The transgenic embryonic fibroblasts survive under a wide range of histidinol-containing growth conditions and support growth of ES cell cultures.     

Chondrocyte cytokine and growth factor expression in murine osteoarthritis

Eighty-five percent of male STR/ort mice develop osteoarthritic lesions of the knee joint by 35 weeks of age. We have developed a non-radioactive in-situ hybridization method using digoxigenin-labeled oligonucleotide probes to study the expression of the cytokines interleukin (IL) 1 alpha, Il-1 beta and IL-6 and the growth factors insulin-like growth factor-1 (IGF-1) and transforming growth factor beta (TGF beta 1) during the development of osteoarthritis (OA) in this model. Age- and sex-matched CBA mice, which do not develop OA, showed no detectable expression of any of the cytokines or growth factors studied. In contrast, 20-week-old STR/ort mice with no OA lesions showed positive expression [positive: (+)] for all the cytokines and growth factors studied. At 35 weeks of age, STR/ort mice with varying grades of OA showed positive (+) or strong (++) signals for both cytokines and growth factors throughout the tibial articular cartilage. The strongest signal was seen in areas where OA lesions were present. In areas of histologically-normal cartilage adjacent to the lesions, the signals were still positive but weaker. Fifty-week-old STR/ort mice with OA lesions showed a similar pattern of expression to 35-week-old mice. Thirty-five or 50-week-old STR/ort mice with no OA lesions had much reduced expression compared with those with OA lesions. These mice may be indicative of those STR/ort mice which do not develop OA. The results seen in the STR/ort together with previous biochemical analyses are consistent with an up-regulation of anabolic growth factors and catabolic cytokines in the prelesional stages of OA with anabolic effects predominating. At later stages of OA, the effects of catabolic factors appear to predominate and osteoarthritic lesions become evident.     

Serotonin blocks different patterns of low Mg2+-induced epileptiform activity in rat entorhinal cortex, but not hippocampus

Low Mg2+-induced epileptiform activity in the entorhinal cortex is characterized by an initial expression of seizure-like events followed by late recurrent discharges. Both these forms of activity as well as the transition between them were blocked by serotonin. In contrast, serotonin had little effect upon the epileptiform activity in areas CA3 and CA1 of the hippocampus. Both forms of epileptiform activity in the entorhinal cortex are sensitive to N-methyl-D-aspartate receptor antagonists and it is shown here that serotonin blocked both types of epileptiform activity through an effective concentration-dependent reduction of N-methyl-D-aspartate receptor-mediated excitatory postsynaptic potentials in deep layer entorhinal cortex cells. Serotonin also prolonged or even prevented the transition between the two types of epileptiform activity and we suggest that this may be through activation of the Na+/K+-ATPase. The resistance of epileptiform activity in CA1 and CA3 to serotonin was most likely related to the inability of serotonin to reduce Schaffer collateral-evoked excitatory postsynaptic potentials. Given the strong serotonergic inputs to both the hippocampus and entorhinal cortex, the differential sensitivity of the two regions to serotonin suggests functional differences. In addition since the late recurrent discharges in the entorhinal cortex are resistant to all clinically used anticonvulsants, serotonin may open new avenues for the development of novel anticonvulsant compounds.     

Wiskott-Aldrich syndrome/X-linked thrombocytopenia: WASP gene mutations, protein expression, and phenotype

Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT), caused by mutations of the WAS protein (WASP) gene, represent different phenotypes of the same disease. To demonstrate a phenotype/genotype correlation, we determined WASP gene mutations in 48 unrelated WAS families. Mutations included missense (20 families) and nonsense (eight) mutations located mostly in exons 1 to 4, and splice-site mutations (seven) and deletions and insertions (13) located preferentially in exons 7 to 11. Both genomic DNA and cDNA were sequenced and WASP expression was measured in cell lysates using peptide-specific rabbit anti-WASP antibodies. WASP was expressed in hematopoietic cell lines including bone marrow-derived CD34+ cells. Missense mutations located in exons 1 to 3 caused mild disease in all but one family and permitted WASP expression, although frequently at decreased concentration. Missense mutations affecting exon 4 were associated with classic WAS and, with one exception, barely detectable WASP. Nonsense mutations caused classic WAS and lack of protein. Insertions, deletions, and splice-site mutations resulted in classic WAS and absent, unstable, truncated, or multiply spliced protein. Using affinity precipitation, WASP was found to bind to Src SH3-containing proteins Fyn, Lck, PLC-gamma, and Grb2, and mutated WASP, if expressed, was able to bind to Fyn-glutathione S-transferase (GST) fusion protein. We conclude that missense mutations affecting the PH domain (exons 1 to 3) of WASP inhibit less important functions of the protein and result in a mild phenotype, and that missense mutations affecting exon 4 and complex mutations affecting the 3' portion of WASP interfere with crucial functions of the protein and cause classic WAS.