The effect of cryotherapy on total antioxidative capacity in patients with active seropositive rheumatoid arthritis.

Patients with rheumatoid arthritis (RA) have increased oxidative stress, decreased antioxidant levels, and impaired antioxidant capacity. Cold treatments are used to relieve joint inflammation and pain. Therefore, we measured the effect of cold treatments on the antioxidative capacity of RA patients with active disease. Sixty patients were randomized to (1) whole body cryotherapy at -110 °C, (2) whole body cryotherapy at -60 °C, or (3) local cryotherapy. Each treatment was given three times daily for 7 consecutive days in addition to the conventional rehabilitation. Blinded rheumatologist evaluated disease activity before the first and after the last cryotherapy. We collected plasma samples daily immediately before the first and after the second cryotherapy and measured total peroxyl radical trapping antioxidant capacity of plasma (TRAP), which reflects global combined antioxidant capacity of all individual antioxidants in plasma. Baseline morning TRAP levels (mean, 95% CI), adjusted for age, body mass index, disease activity, and dose of prednisolone, were 1244 (1098-1391) µM/l in the local cryotherapy, 1133 (1022-1245) µM/l in the cryotherapy at -60 °C, and 989 (895-1082) µM/l in the cryotherapy at -110 °C groups (p = 0.006). After the first treatment, there was a rise in 1-h TRAP of 14.2 (-4.2 to 32.6) µM/l, 16.1 (-7.4 to 39.6) µM/l, and 23.6 (4.1-43.2) µM/l, respectively. The increase was significant in the whole-body cryotherapy -110 °C group (p < 0.001) but not significant between the groups (p = 0.78). When analyzed for the whole week, the daily morning TRAP values differed significantly between the treatment groups (p = 0.021), but there was no significant change within each treatment group. Whole-body cryotherapy at -110 °C induced a short-term increase in TRAP during the first treatment session with but not during other treatment modalities. The effect was short and the cold treatments did not cause a significant oxidative stress or adaptation during 1 week.

Acute cold stress in rheumatoid arthritis inadequately activates stress responses and induces an increase of interleukin 6.

OBJECTIVE: Acute stress in patients with rheumatoid arthritis (RA) should stimulate a strong stress response. After cryotherapy, we expected to observe an increase of hormones of the adrenal gland and the sympathetic nervous system. METHODS: A total of 55 patients with RA were recruited for whole-body cryotherapy at -110 degrees C and -60 degrees C, and local cold therapy between -20 degrees C and -30 degrees C for 7 days. We measured plasma levels of steroid hormones, neuropeptide Y (sympathetic marker), and interleukin (IL)6 daily before and after cryotherapy. RESULTS: In both therapy groups with/without glucocorticoids (GC), hormone and IL6 levels at baseline and 5 h after cold stress did not change over 7 days of cryotherapy. In patients without GC, plasma levels of cortisol and androstenedione were highest after -110 degrees C cold stress followed by -60 degrees C or local cold stress. The opposite was found in patients under GC therapy, in whom, unexpectedly, -110 degrees C cold stress elicited the smallest responses. In patients without GC, adrenal cortisol production increased relative to other adrenal steroids, and again the opposite was seen under GC therapy with a loss of cortisol and an increase of dehydroepiandrosterone. Importantly, there was no sympathetic stress response in both groups. Patients without GC and -110 degrees C cold stress demonstrated higher plasma IL6 compared to the other treatment groups (not observed under GC), but they showed the best clinical response. CONCLUSIONS: We detected an inadequate stress response in patients with GC. It is further shown that the sympathetic stress response was inadequate in patients with/without GC. Paradoxically, plasma levels of IL6 increased under strong cold stress in patients without GC. These findings confirm dysfunctional stress axes in RA.

Expression of mRNAs coding for the alpha 1 chain of type XIII collagen in human fetal tissues: comparison with expression of mRNAs for collagen types I, II, and III.

This paper describes the topographic distribution of the multiple mRNAs coding for a novel human short-chain collagen, the alpha 1 chain of type XIII collagen. To identify the tissues and cells expressing these mRNAs, human fetal tissues of 15-19 gestational wk were studied by Northern and in situ hybridizations. The distribution pattern of the type XIII collagen mRNAs was compared with that of fibrillar collagen types I, II, and III using specific human cDNA probes for each collagen type. Northern hybridization showed the bone, cartilage, intestine, skin, and striated muscle to contain mRNAs for type XIII collagen. An intense in situ hybridization signal was obtained with the type XIII collagen cDNAs in the epidermis, hair follicles, and nail root cells of the skin, whereas the fibrillar collagen mRNAs were detected in the dermis. Cells in the intestinal mucosal layer also appeared to contain high levels of alpha 1(XIII) collagen mRNAs, but contained none of the fibrillar collagen mRNAs. In the bone and striated muscle, alpha 1(XIII) collagen mRNAs were detected in the mesenchymal cells forming the reticulin fibers of the bone marrow and endomycium. The hybridization signal obtained with the alpha 1(XIII) collagen cDNA probe in cartilaginous areas of the growth plates was similar, but less intense, to that obtained with the type II collagen probe. A clear hybridization signal was also detected at the (pre)articular surfaces and at the margins of the epiphyses, whereas it was weaker in the resting chondrocytes in the middle of the epiphyses. The brain, heart, kidney, liver, lung, placenta, spleen, testis, tendon, and thymus did not appear to contain alpha 1(XIII) collagen mRNAs.

The N-myc proto-oncogene and IGF-II growth factor mRNAs are expressed by distinct cells in human fetal kidney and brain.

We studied the expression of the N-myc proto-oncogene and the insulin-like growth factor-II (IGF-II) gene in human fetuses of 16-19 gestational wk. Both genes have specific roles in the growth and differentiation of embryonic tissues, such as the kidney and neural tissue. Since continued expression of N-myc and IGF-II mRNAs is also a characteristic feature of Wilms' tumor, a childhood neoplasm of probable fetal kidney origin, we were particularly interested in the possibility that their expression might be linked or coordinately regulated in the developing kidney. Expression of N-myc mRNA was observed in the brain and in the kidney by Northern hybridization analysis. In in situ hybridization of the kidney, N-myc autoradiographic grains were primarily located over epithelially differentiating mesenchyme while most of the mesenchymal stromal cells showed only a background signal with the N-myc probe. N-myc mRNA was detectable throughout the developing brain with a slight accentuation in the intermediate zone cells in between the subependymal and cortical layers. Thus, even postmitotic neuroepithelial cells of the fetal cerebrum expressed N-myc mRNA. In Northern hybridization, IGF-II mRNA signal was abundant in the kidney but much weaker, though definite, in the brain. The regional distribution of IGF-II mRNA in the kidney was largely complementary to that of N-myc. IGF-II autoradiographic grains were located predominantly over the stromal and blastemal cells with a relative lack of hybridization over the epithelial structures. In the brain, IGF-II mRNA was about two- to threefold more abundant in the subependymal and intermediate layers than in the cortical plate and ependymal zone, respectively. The fetal expression patterns of the N-myc and IGF-II mRNAs are reflected by the types of tumors known to express the corresponding genes during postnatal life such as Wilms' tumor. However, the apparent coexpression of the IGF-II and N-myc genes in immature kidneys occurs largely in distinct cell types.

A truncated laminin chain homologous to the B2 chain: structure, spatial expression, and chromosomal assignment.

We describe the identification of a novel laminin chain. Overlapping clones were isolated from a human fibrosarcoma HT1080 cell cDNA library spanning a total of 5,200 bp. A second set of clones contained an alternative 3' end sequence giving a total of 4,316 bp. The longer sequence contained an open reading frame for a 1,193-residue-long polypeptide. The alternative sequence was shortened at the carboxyl-terminal end coding for a 1,111-residue-long polypeptide. The amino acid sequence contained 21 amino acids of a putative signal peptide and 1,172 residues or alternatively 1,090 residues of a sequence with five distinct domains homologous to domains I-V in laminin chains. Comparison of the amino acid sequences showed that the novel laminin chain is homologous to the laminin B2 chain. However, the structure of the novel laminin chain isolated here differs significantly from that of the B2 chain in that it has no domain VI and domains V, IV, and III are shorter, resulting in a truncated laminin chain. The alternative sequence had a shortened domain I/II. In accordance with the current nomenclature, the chain characterized here is termed B2t. Calculation of possible chain interactions of laminin chains with the B2t chain domain I/II indicated that the B2t chain can replace the B2 chain in some laminin molecules. The gene for the laminin B2t chain (LAMB2T) was localized to chromosome 1q25-q31 in close proximity to the laminin B2 chain gene. Northern analysis showed that the B2t chain is expressed in several human fetal tissues but differently from the laminin B1 and B2 chains. By in situ hybridization expression of the B2t chain was localized to specific epithelial cells in skin, lung, and kidney as opposed to a general epithelial and endothelial cell expression of the laminin B2 chain in the same tissues.

Human laminin M chain (merosin): complete primary structure, chromosomal assignment, and expression of the M and A chain in human fetal tissues.

The primary structure of the human laminin M chain was determined from cDNA clones isolated from human placental libraries. The clones covered a total of 6,942 bp, with 49-bp encoding a 5' end untranslated region and 6,893-bp coding for a translated sequence. The complete human laminin M chain contains a 22-residue signal peptide and 3,088 residues of the mature M chain. The M chain has a domain structure similar to that of the human and mouse A chains. The homology between the two human laminin heavy chains is highest in the short arm region and lowest in the long arm helical domain I + II. Northern blot analysis of human fetal tissues showed that the M chain was expressed in most tissues such as cardiac muscle, pancreas, lung, spleen, kidney, adrenal gland, skin, testis, meninges, choroid plexus, and some other regions of the brain, but not in liver, thymus, and bone. In situ hybridization localized the expression of the M chain gene to cells of mesenchymal origin. In contrast, expression of the A chain was observed only in kidney, testis, neuroretina and some region of brain as determined by Northern analyses. Epithelial and endothelial cells were negative for both M and A chain gene transcripts. The gene for the human M chain (LAMM) was localized to chromosome 6q22-->23.

Effectiveness of different cryotherapies on pain and disease activity in active rheumatoid arthritis. A randomised single blinded controlled trial.

OBJECTIVE: Local cryotherapy is used to relieve pain and inflammation in injuries and inflammatory conditions. Whole-body cryotherapy is an extreme method administered at -110 degrees C for 2 to 3 minutes. The aim of the study was to compare the effect of cryotherapies on pain and inflammation in patients with rheumatoid arthritis (RA). METHODS: Sixty patients with active seropositive RA were recruited in a randomised controlled single-blinded study to receive whole-body cryotherapy at -110 degrees C, whole-body cryotherapy at -60 degrees C, application of local cold air at -30 degrees C and the use of cold packs locally. In the final analysis, the last 2 groups were pooled. The patients had 2-3 cryotherapy sessions daily for one week plus conventional physiotherapy. Clinical and laboratory variables and patient's and physician's global assessments were used to assess the outcome. Disease activity was calculated by DAS. RESULTS: Pain decreased in all treatment groups, most markedly in the whole-body cryotherapy (-110 degrees C) group. DAS decreased slightly with no statistically significant differences between the groups. No serious or permanent adverse effects were detected. Six of 40 patients (15%) discontinued the whole-body cryotherapy. CONCLUSION: Pain seemed to decrease more in patients in the whole-body cryotherapy at -110 degrees C than during other cryotherapies, but there were no significant differences in the disease activity between the groups. However, cryotherapy at -110 degrees C is expensive and available only in special centres and may have minor adverse effects. Based on our results, whole-body cryotherapy at -110 degrees C is not superior to local cryotherapy commonly used in RA patients for pain relief and as an adjunct to physiotherapy.

Expression of the myc proto-oncogenes in developing human fetal brain.

We have analysed c-myc, N-myc and L-myc gene expression in developing human fetal brain by Northern hybridization, RNAase protection and in situ hybridization. The unique zonal organization of the developing fetal brain allows a particularly good assessment of the coupling of myc gene expression to cell proliferation and differentiation in vivo. By Northern and in situ hybridization, L-myc as well as c-myc and N-myc transcripts in the brain were found in the post-mitotic cortical and intermediate layers, as well as in the mitotically active layers containing the neuroepithelial precursor cells. Consistent results were also obtained for L-myc using RNAase protection analysis. Both the 3.6 and 3.8kb forms of the L-myc mRNA, resulting from alternative splicing of intron I, were detected in layers of neuroectodermal origin, but not in the meninges or choroid plexus. We also extended L-myc expression and splicing analyses to other developing human fetal tissues. L-myc mRNA was expressed in several other fetal tissues, particularly in fetal skin. Predominantly intron I containing L-myc mRNA was observed in fetal striated and cardiac muscle. Thus, L-myc is expressed in a wider spectrum of developing tissues than previously known. Our findings also, show that L-myc as well as N-myc and c-myc expression is uncoupled from cell division in developing brain.

cyl encodes a putative cytoplasmic tyrosine kinase lacking the conserved tyrosine autophosphorylation site (Y416src).

We have isolated a cDNA encoding a novel tyrosine kinase family member, named cyl (consensus tyrosine-lacking kinase), from the K562 human leukemia cell line. The deduced cyl protein lacks signal and transmembrane sequences but contains features of known cytoplasmic tyrosine kinases, including amino-terminal SH3 and SH2 domains. However, having very short amino and carboxy termini, cyl does not seem to belong to any of the previously characterized subfamilies of cytoplasmic tyrosine kinases. Furthermore, cyl lacks the highly conserved tyrosine autophosphorylation site (Y416src) in the tyrosine kinase catalytic domain. The cyl gene is located on human chromosome 15. It is expressed ubiquitously as two independently regulated mRNA species of 2.6 and 3.4 kb in human leukemia cell lines and fetal tissues.

The rearranged L-myc fusion gene (RLF) encodes a Zn-15 related zinc finger protein.

We have previously characterized intrachromosomal rearrangements at 1p32 fusing the first exon of the RLF gene with L-myc. Here we present the full-length cDNA sequence of the 6251 bp RLF mRNA. The predicted 1914 amino acid Rlf protein contains sixteen widely spaced zinc finger motifs, and is related to the Zn-15 transcription factor. RLF is widely expressed in fetal and adult tissues, suggesting that it has a general role in transcriptional regulation. The zinc fingers are not contained in the 79 amino acid N-terminal region of RLF involved in the RLF-L-myc fusions, and the transforming ability of the RLF-L-myc and the normal L-myc proteins is indistinguishable. These findings suggest that the role of the rearrangements fusing RLF and L-myc is to deregulate the tightly controlled expression of the L-myc gene.

Tie endothelial cell-specific receptor tyrosine kinase is upregulated in the vasculature of arteriovenous malformations.

Arteriovenous malformations (AVMs) are congenital lesions composed of abnormal vasculature, with no capillary component, and are clinically significant due to their tendency to spontaneously hemorrhage. The mechanisms regulating the genesis and progression of these lesions are unknown. In order to study the role of angiogenesis in AVMs, we have analyzed the expression of the endothelial cell mitogen vascular endothelial growth factor (VEGF) and a novel endothelial cell-specific receptor tyrosine kinase, Tie, by in situ hybridization and immunohistochemistry in these malformations and surrounding brain tissue. We have previously shown upregulation of Tie accompanying wound healing and tumor progression. In this study, we demonstrate significantly elevated levels of Tie mRNA and protein in AVM and surrounding brain vasculature. Upregulation of VEGF mRNA was observed in the cells of brain parenchyma adjacent to the AVM, and VEGF protein was detected in this tissue as well as in AVM endothelia. Normal brain, in comparison, expressed little or no Tie or VEGF. The significant upregulation of VEGF and Tie in AVMs may indicate some ongoing angiogenesis, possibly contributing to the slow growth and maintenance of the AVM, and could be of potential use in the therapeutic targeting of these lesions.

Effects of paclitaxel with or without cremophor EL on cellular clonogenic survival and apoptosis.

Paclitaxel is currently formulated in a vehicle of 50% ethanol and 50% polyethoxylated surfactant cremophor EL. Cremophor EL has been reported to reverse P-glycoprotein-mediated multidrug resistance (MDR) at doses which are clinically achievable. It has also been reported to have a cytotoxic effect per se. In this study we used two different methods to evaluate the survival of cells exposed to paclitaxel with or without cremophor EL and the vehicle alone. Two laryngeal SCC cell lines (UT-SCC-19A and UT-SCC-29) and two ovarian adenocarcinoma cell lines (UT-OC-3 and UT-OC-5) established in our laboratory were investigated. Northern hybridisation was used to study the mdr-1 mRNA expression of the cell lines. With sensitive Northern analyses, these four lines yielded mdr-1 mRNA signals of the expected 4.5 kb size and of variable intensity, generally at higher levels than those in the positive control cell line KB. The 96-well plate clonogenic assay was used to obtain the fraction survival data and apoptosis was recorded by time-lapse video microscopy. Both methods indicate that cremophor EL alone has no effect on cellular survival. Consequently, paclitaxel without cremophor EL is as active as paclitaxel with cremophor EL in vitro.

Differential expression of two alpha 2-adrenergic receptor subtype mRNAs in human tissues.

Genetic subtypes of alpha 2-adrenergic receptors (AR) may mediate distinct physiological functions, and undergo differential cell type-specific regulation. Thus, these distinct receptor subtypes are possible targets for the development of subtype-selective drugs. We have analyzed the tissue distribution of two human alpha 2-adrenoceptor subtype gene mRNAs, alpha 2-C4 and alpha 2-C10, in normal human fetal and adult tissues. Both receptor subtype mRNAs were abundantly expressed in fetal brain and choroid plexus. In non-neural fetal tissues, alpha 2-C10 mRNA was detected in spleen, kidney, adrenal gland, and skin, while alpha 2-C4 transcripts were observed only in kidney and skin. Most regions of the adult brain also expressed both subtypes, but with marked quantitative differences. For example, cerebral cortex contained predominantly alpha 2-C10 mRNA, whereas the caudate nucleus expressed mostly alpha 2-C4 mRNA. In adult peripheral tissues, alpha 2-C10 mRNA expression was most abundant in spleen and renal cortex, and expression of alpha 2-C4 mRNA was strongest in renal cortex and medulla. These different expression patterns provide evidence for the differential regulation of the two alpha 2-adrenergic receptor genes and warrant further investigation with techniques capable of improved anatomical resolution. Regional differences in receptor subtype expression may be valuable for the development of new, subtype-selective pharmacological agents with more targeted actions compared to currently used alpha 2-adrenoceptor agonists and antagonists.

L-myc and N-myc in hematopoietic malignancies.

The myc proto-oncogenes encode nuclear DNA-binding phosphoproteins which regulate cell proliferation and differentiation. The c-myc gene is implicated in hematopoietic malignancies on the basis of its frequent deregulation in naturally occurring leukemias and lymphomas. Recent evidence suggests that also the N-myc and L-myc genes may have a role in normal and malignant hematopoiesis. N-myc and to a certain degree L-myc can substitute for c-myc in transformation assays in vitro, and their overexpression can block the differentiation of leukemia cell lines. Immunoglobulin heavy chain enhancer (IgH) -driven overexpression of N-myc or L-myc genes cause lymphatic and myeloid tumors, respectively, in transgenic mice. Furthermore, the L-myc and N-myc genes are expressed in several human leukemias and leukemia cell lines, L-myc predominantly in myeloid and N-myc both in myeloid and in some lymphoid leukemias. All N/L-myc positive leukemias and leukemia cell lines coexpress the c-myc gene, thus exemplifying a lack of negative cross-regulation between the different myc genes in leukemia cells. Taken together, these data suggest that L-myc and N-myc may participate in the growth regulation of hematopoietic cells.

[Efficiency of the blood cell separator Haemonetic 30 in the separation of platelets from single donors]. / Leistungsfähigkeit des Blutzellseparators Haemonetic 30 bei der Plättchengewinnung aus Einzelspendern.

By means of the Haemonetic 30 separator we were able to collect 4.1 +/- 0.94 X 10(11) (X +/- 1 SD) platelets in 57 procedures consisting of 5-6 cycles. The platelet yield was significantly influenced by the blood volume processed and by initial platelet counts in donors. Augmenting platelet collection volume in the red colored part of the buffy-coat from 10-20 ml did not increase the yield. Transfusion of collected platelets into 27 non-sensitized patients showed normal recovery of 43% at one hour and shortening of bleeding time from over 30 min to a mean of 5 min 20 sec in 12 patients tested. Autotransfusion of 51Cr-labeled platelets into 8 healthy donors revealed identical recoveries (42 +/- 12%) and a normal survival time (9.2 +/- 0.7 days).

Genomic variation of herpes simplex virus type 2 isolates analysed by hybridization after electroblotting from polyacrylamide gels.

Genomic variation of herpes simplex virus type 2 (HSV-2) strains was analysed by polyacrylamide gradient gel electrophoresis and subsequent hybridization to cloned HSV-2 sequences. Two probes were used, one from the L-segment and one from the S-segment of the HSV-2 genome. The probes did not contain a-repeat sequences. Hybridization to the specific sequences in individual DNA fragments obtained by use of the frequently cleaving restriction endonuclease Alu I revealed variations in the genome not detectable by analysing the fragment size only. The use of 35S-labelled deoxynucleotide in the radioactive labelling of the probe further improved the resolution of the method.