Effect of topical treatment with 7.5% urea in Ichthyosis Vulgaris: A randomized, controlled, double blinded, split body study evaluating the effect of urea cream compared to the vehicle (moisturizing) cream.

Background: Ichthyosis Vulgaris (IV) is a common genetic skin disease, characterized by dry, scaling skin and itch. Urea cream has been a central part of IV treatment for decades, but only few studies have evaluated the effect of urea compared to basic moisturizers. Objective: To evaluate the treatment effect of 7.5% urea cream compared to a basic moisturizer in patients with IV. Methods: Participants (n = 14) were randomized to apply the 7.5% urea cream on one body half and a basic moisturizer on the other during a study period of 4 weeks. Measuring points on participants arms and legs were evaluated at baseline and at endpoint with a patient questionnaire visual assessment scale (VAS), a clinical scoring, and electronic skin hydration analysis to assess the treatment effects. Results: On the arms, no significant differences between the two treatments were found. On the legs, however, the urea treated areas had a significantly higher decrease in SRRC score (0.7 points [95% CI: 1.1-0.3, p < 0.005]) and increase in hydration (32.1 µS [95% CI: 10.9-53.2, p < 0.006]). Conclusion: Skin hydration improved significantly with both urea and moisturising treatment. On the legs, with most keratinization, urea was superior.Trial registry: https://clinicaltrials.gov/ct2/show/NCT02978209?cond=Ichthyosis+Vulgaris&draw=1&rank=2.

A novel mutation in the connexin 26 gene (GJB2) in a child with clinical and histological features of keratitis-ichthyosis-deafness (KID) syndrome.

BACKGROUND: Keratitis-ichthyosis-deafness (KID) syndrome is a rare congenital ectodermal disorder, caused by heterozygous missense mutation in GJB2, encoding the gap junction protein connexin 26. The commonest mutation is the p.Asp50Asn mutation, and only a few other mutations have been described to date. AIM: To report the fatal clinical course and characterize the genetic background of a premature male neonate with the clinical and histological features of KID syndrome. METHODS: Genomic DNA was extracted from peripheral blood and used for PCR amplification of the GJB2 gene. Direct sequencing was used for mutation analysis. RESULTS: The clinical features included hearing impairment, ichthyosiform erythroderma with hyperkeratotic plaques, palmoplantar keratoderma, alopecia of the scalp and eyelashes, and a thick vernix caseosa-like covering of the scalp. On histological analysis, features characteristic of KID syndrome, such as acanthosis and papillomatosis of the epidermis with basket-weave hyperkeratosis, were seen. The skin symptoms were treated successfully with acitretin 0.5 mg/kg. The boy developed intraventricular and intracerebral haemorrhage, leading to hydrocephalus. His condition was further complicated by septicaemia and meningitis caused by infection with extended-spectrum beta-lactamase-producing Klebsiella pneumoniae. Severe respiratory failure followed, and the child died at 46 weeks of gestational age (13 weeks postnatally). Sequencing of the GJB2 gene showed that the child was heterozygous for a novel nucleotide change, c.263C>T, in exon 2, leading to a substitution of alanine for valine at position 88 (p.Ala88Val). CONCLUSIONS: This study has identified a new heterozygous de novo mutation in the Cx26 gene (c.263C>T; p.Ala88Val) leading to KID syndrome.

Natural killer cell-mediated basal and interferon-enhanced cytotoxicity against liver cancer cells is significantly impaired under in vivo oxygen conditions.

Current immunostimulatory treatment protocols of cancer are often met with little success. Several lines of evidence indicate that the tumour microenvironment may impair the cytotoxic activity of natural killer (NK) cells. In this study, the NK cell-mediated killing of liver-derived cells was investigated at oxygen concentrations conform to those present in the human body at physiological and pathological conditions. The in vivo-relevant oxygen concentrations corresponding to 1, 2 and 6% were compared to those of the ambient air (21%) for their effect on the lysis of four liver-derived cell lines and the highly susceptible K562 cells. Exposure to each of the hypoxic conditions had a significantly inhibitory effect on NK cytotoxicity. Treatment with interferon-alpha (IFN-alpha) in hypoxia enhanced the cytotoxic potential of the NK cells less than it enhanced the cytotoxicity at ambient oxygen conditions. In summary, the oxygen tension profoundly affects both the cytoxic activity of NK cells and their activation by IFN-alpha.

Chemokine receptor CCR2b 64I polymorphism and its relation to CD4 T-cell counts and disease progression in a Danish cohort of HIV-infected individuals. Copenhagen AIDS cohort.

We have investigated the role of the recently described mutation in CCR2b named 64I in relation to HIV resistance, CD4 T-cell counts, and disease progression in Danish individuals by polymerase chain reaction (PCR)-based methods as well as sequenced full-length CXCR4 and CCR5 genes from HIV-infected long-term nonprogressors for possible mutations. In total, 215 Danish individuals were analyzed for 64I allele frequency; disease progression was followed in 105 HIV-1-positive homosexual Danish men from their first known positive HIV-1 test result and up to 11 years. In 87 individuals, the CD4 T-cell count was monitored closely. We found no significant difference in 64I allele frequency between HIV-1-seropositive persons (0.08), high-risk HIV-1-seronegative persons (0.11), and blood donors (0.06). No significant difference was observed in annual CD4 T-cell decline, CD4 T-cell counts at the time of AIDS, in AIDS-free survival as well as survival with AIDS, between 64I allele carriers and wild-type individuals. Among 9 long-term nonprogressors, 2 carried the 64I allele, while none of 9 fast progressors carried the 64I allele. However, this was not significantly different (p=.47). Long-term nonprogression could not be explained by CXCR4 polymorphism or other polymorphisms in the CCR5 gene than the CCR5delta32 allele. Furthermore, we were not able to detect any significant independent effect of the 64I allele on development to AIDS, overall survival, and annual CD4 T-cell decline in this cohort.

HIV-infected individuals with the CCR delta32/CCR5 genotype have lower HIV RNA levels and higher CD4 cell counts in the early years of the infection than do patients with the wild type. Copenhagen AIDS Cohort Study Group.

The relations among serum HIV RNA levels, CD4 cell counts, presence of the mutant CCR5-allele in heterozygous form, and clinical outcome was analyzed in 96 patients from the Copenhagen AIDS Cohort. In the early years of the infection, patients with the CCR5 delta32/CCR5 genotype had significantly lower HIV RNA levels (p = 0.005) and higher CD4 cell counts (p < 0.005) than did patients homozygous for the normal allele. The long-term clinical benefit of being heterozygous is small and cannot solely explain the large interpatient variation in progression rates. The beneficial effect of being heterozygous seems to be mediated by events in the early stages of the HIV infection.

Efficient in vitro inhibition of HIV-1 gag reverse transcription by peptide nucleic acid (PNA) at minimal ratios of PNA/RNA.

We have tested the inhibitory potential of peptide nucleic acid (PNA) on in vitro reverse transcription of the HIV-1 gag gene. PNA was designed to target different regions of the HIV-1 gag gene and the effect on reverse transcription by HIV-1, MMLV and AMV reverse transcriptases (RTs) was investigated. We found that a bis-PNA (parallel antisense 10mer linked to antiparallel antisense 10mer) was superior to both the parallel antisense 10mer and antiparallel antisense 10mer in inhibiting reverse transcription of the gene, thus indicating triplex formation at the target sequence. A complete arrest of reverse transcription was obtained at approximately 6-fold molar excess of the bis-PNA with respect to the gag RNA. At this molar ratio we found no effect on in vitro translation of gag RNA. A 15mer duplex-forming PNA was also found to inhibit reverse transcription at very low molar ratios of PNA/ gag RNA. Specificity of the inhibition of reverse transcription by PNA was confirmed by RNA sequencing, which revealed that all tested RTs were stopped by the PNA/RNA complex at the predicted site. We propose that the effect of PNA is exclusively due to steric hindrance, as we found no signs of RNA degradation that would indicate PNA-mediated RNase H activation of the tested RTs. In conclusion, PNA appears to have a potential to become a specific and efficient inhibitor of reverse transcription in vivo , provided sufficient intracellular levels are achievable.

Heterozygosity for a deletion in the CKR-5 gene leads to prolonged AIDS-free survival and slower CD4 T-cell decline in a cohort of HIV-seropositive individuals.

OBJECTIVE: Recently, it has been shown that a homozygous 32 base-pair deletion in the gene encoding CKR-5, a major coreceptor for HIV-1, leads to resistance to infection with HIV-1. We have investigated whether HIV-seropositive individuals who were heterozygous for the CKR-5 deletion had a different course of the disease. DESIGN: Thirty-five high-risk HIV-seronegative and 99 HIV-seropositive Danish homosexual men followed form 1985 to 1996 and 37 blood donors were analysed for their CKR-5 genotype by polymerase chain reaction. RESULTS: Two (6%) of the 35 HIV-seropositive subjects at high-risk of infection were homozygous and seven (20%) were heterozygous for the CKR-5 deletion. This was not significantly different from the distribution in normal donors. Twenty-two (22%) of the 99 HIV-seropositive subjects were heterozygous and none was homozygous. Two subgroups of patients who had an opposite course of the HIV disease were identified. Of nine long-term non-progressors, six (66%) were heterozygous for the deletion. This frequency is significantly higher than in nine rapid progressors of whom non was heterozygous. The frequency of heterozygotes in long-term non-progressors was also significantly higher than in the cohort as a whole. A Kaplan-Meier plot of the HIV-seropositive subjects, of whom 57 developed AIDS, showed a significantly better prognosis within the first 7 years of follow-up for those who were heterozygous for the deletion. Heterozygous individuals also had a significantly slower decrease in CD4 T-cell count per year. CONCLUSION: Individuals who are heterozygous for the 32-base-pair deletion in the CKR-5 gene have a slower decrease in their CD4 T-cell count and a longer AIDS-free survival than individuals with the wild-type gene for up to 11 years of follow-up.

Developmental regulation of tissue transglutaminase during human placentation and expression in neoplastic trophoblast.

The expression of tissue transglutaminase (tTG) was studied during the formation of the normal human placenta and in molar pregnancies and choriocarcinoma, in order to correlate its expression with the functional characteristics of the recognized trophoblast cell types. tTG expression was found to be developmentally regulated. Before 6-7 weeks' gestation, only the chorionic villous cytotrophoblast expresses tTG. Thereafter the overlying syncytiotrophoblast becomes positive. tTG expression is gradually downregulated in the intermediate trophoblast cells emerging from the tips of the chorionic villi invading the uterine tissue. In the decidual wall, the intermediate trophoblast does not express tTG, whereas scattered syncytial cells, the placental bed giant cells, express tTG. Villi from complete hydatidiform mole (CHM) show tTG expression in both the cyto- and the syncytiotrophoblast. The intermediate trophoblast cells from CHM show heterogeneous tTG expression, with a majority of negative cells, whereas extravillous syncytia always express tTG. In choriocarcinoma, the tumour cells show heterogeneous tTG expression, with a majority of positive cells. Analysis of tTG protein and mRNA in placental extracts by Western and Northern blotting did not provide evidence for expression of the truncated form of tTG found in some cell types. The regulated expression of tTG in the normal placenta suggests that the enzyme is involved in important trophoblastic functions and may participate in the control of invasion.

Transfer of human T cell lymphotropic virus type I to human term trophoblast cells in vitro.

We studied the susceptibility of term placental trophoblast cells to in vitro infection with human T cell leukaemia/lymphotropic virus type I in order to provide further insight into the role of syncytiotrophoblast in transplacental passage of the virus. Pure villous trophoblast cultures were exposed to cell-free virus and the extent of infection was analysed by semiquantitative PCR assay to detect integrated proviral DNA. Four different primer pairs targeting the gag, pol, env and pX regions invariably revealed that virus sequences were present in amounts 10(2)-10(3) times less than in the reference cell line MT-2. Virus expression was studied at both the transcriptional and translational levels. Whereas doubly spliced mRNAs coding for the Tax and Rex regulatory proteins could be detected by RT-PCR no virus-specific proteins were found in the cells by immunoperoxidase staining. The present data lend support to the notion that the placental trophoblast may represent a barrier effectively protecting the fetal compartment from exposure to the virus.

Cilia-mediated oriented chemokinesis in Tetrahymena thermophila.

The role of the cilia in the locomotion ("gliding") of Tetrahymena thermophila in a semi-solid medium has been studied when cells were migrating in gradients of attractant. Video recordings and computer-aided motion analysis of migrating cells and their ciliary activity show that Tetrahymena thermophila migrate by swimming forward in semi-solid methyl cellulose, using their cilia. Ciliary reversals occur at certain intervals and cause a termination ("stop") of cellular migration. Cells with reversed cilia resume forward migration when normal ciliary beating resumes. In gradients of attractants, cells migrating towards the attractant suppress ciliary reversals, which leads to longer runs between stops than in control cells. Cells migrating away from the attractant have a higher frequency of ciliary reversals than the control cells resulting in shorter runs. Stimulated cells adapt to a particular ambient concentration of attractant several times during migration in the gradient. Adaptation is followed by de-adaptation, which occurs during the "stop." In the presence of cycloheximide, a strong inhibitor of chemoattraction, the attractant-induced suppression of ciliary reversal is abolished (cells become desensitized to the attractant). It is concluded that Tetrahymena has a short-term memory during adaptation. This is important for the efficiency of migration towards an attractant.

Physiological parameters affecting the chemosensory response of Tetrahymena.

We have investigated the significance of a number of physiological parameters in the preparation of cells for experiments on chemokinesis in Tetrahymena. The study comprises (1) growth state of the cells, (2) composition of the starvation medium, (3) concentration of cells during starvation, (4) oxygen saturation of the starvation medium, (5) temperature during starvation, and (6) starvation period. By controlling the physiological state of the cells, we significantly improved the reproducibility of the results obtained in assays for chemokinesis in Tetrahymena. In short, cells optimal for chemokinesis at an assay temperature of 28 degrees C should be starved from the exponential growth phase in a concentration below 2 x 10(5) cells ml-1 for 10-20 h. The surface-to-volume ratio of the starvation medium--water or Hepes buffer--should be about 5 cm-1 (or more) to ensure more than 95% oxygen saturation of the starvation medium. Maximal chemosensory responses were obtained if the cells were starved at 21 degrees C. The chemokinetic potential of the cells decreased significantly, as did the levels of the ratio of ATP to ADP, if cells were starved at higher temperatures. A tentative correlation between the ATP level in the cells and the chemosensory potential of the cells has been found. We suggest that chemokinesis is a constant quality of Tetrahymena, because we found no sign that prolonged starvation or other changes applied to the cells produced an up-regulation of the chemosensory response. Apparently, starvation is obligatory only to remove the growth medium (which is itself a very potent attractant), thereby making the cells sensitive to the chemoattractants.

An improved quantitative assay for chemokinesis in Tetrahymena.

This paper presents a quantitative and sensitive assay for the measurement of chemosensory behavior in Tetrahymena. The two-phase assay is easy to perform in large quantities, so a variety of compounds can be screened under comparable conditions. A suspension of 2 x 10(5) cells ml-1 (the upper phase) is starved for 20-40 h and then gently placed on top of a 5% solution of Metrizamide (the lower phase) in a disposable microcuvette. The optical density of the lower phase is monitored at 600 nm with an automated spectrophotometer at selected time points. Optimum sensitivity of the assay is achieved when the cells slowly but continuously enter the lower phase, so that about 5% of them will be in the lower phase within 30 min. Optimal chemosensory responses occurred in Tetrahymena thermophila at about 25 degrees C. The response was delayed at 15 degrees C and markedly reduced at 35 degrees C. The data suggest three bases for quantifying the response in the assay: (1) initial slope of the absorbance versus time; (2) final maximal absorbance within the time period of measurement; and (3) signal-to-noise ratio (S/N) at a fixed time. We have quantified--in terms of S/N--the chemosensory responses in Tetrahymena for the following compounds: beta-endorphin, fibroblast growth factor, insulin, and platelet-derived growth factor (PDGF); these substances were active in nanomolar concentrations, and the maximal S/N was between 3 and 5.1. Acetylcholine was active only in millimolar concentrations; maximal S/N was 4.1 at 1 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of dividing and non-dividing Tetrahymena cells at different physiological states.

Eight defined physiological states of Tetrahymena pyriformis are described. For dividing cells the states comprise: 1. Exponentially growing cells, 2. Cells at late exponential growth phase, 3. Cells kept at a high cell concentration, 4. Cells shifted up or down by change of medium, temperature or degree of aeration. For non-dividing cells the states are: 5. Cells at stationary phase, 6. Cells during starvation, 7. Cells during shift-up after long-term starvation, 8. Cells at self-induced hypoxia. The different cellular states are described by one or more of the following characteristics: growth rate, volume, swimming speed, oxygen consumption and by the oxygen saturation and the pH in the medium. The results show that T. pyriformis grows equally well in proteose-peptone (PY) medium from 1 cell ml(-1) to 10(3) cells ml(-1) as from - e.g. - 10(2) to 10(5) cells ml(-1). The maximum cell concentration obtained depends on the medium and the availability of oxygen. At shift-down by decrease of temperature the cells grow slower and obtain a considerable oversize. Single cells tolerate starvation for 12 days. The cell volume (electronically determined) decreased from about 7000 µm(3) to about 200 µm(3). Long-term starved cells may be upshifted. Thereby growth without cell division can be studied until the cell volume approaches 2100 µm(3) which is the minimum volume of division competence. Under certain conditions cells may grow into self-induced hypoxia leading to growth arrest. These cells will attain an oversize. The swimming speed at 28°C of exponentially growing cells is 0.33 to 0.59 mm sec(-1) depending on the medium. At lower temperature the swimming speed is decreased. In PY-medium the values are: 28°C (0.57), 16°C (0.50), 9°C (0.37). During starvation the swimming speed decreases from about 0.6 to about 0.1 mm sec(-1) (after 6 days). The oxygen consumption is for state 1 cells: 3.9 µl O(2)/10(6) cells min(-1) (maximal value). The value of hypoxic cultures is 2.1, for cells kept at high concentration 0.4, and for starved cells (24 h) 0.2.