Depression, anxiety and PTSD symptoms before and during the COVID-19 pandemic in the UK.

BACKGROUND: The impact of the coronavirus disease 2019 (COVID-19) pandemic on mental health is still being unravelled. It is important to identify which individuals are at greatest risk of worsening symptoms. This study aimed to examine changes in depression, anxiety and post-traumatic stress disorder (PTSD) symptoms using prospective and retrospective symptom change assessments, and to find and examine the effect of key risk factors. METHOD: Online questionnaires were administered to 34 465 individuals (aged 16 years or above) in April/May 2020 in the UK, recruited from existing cohorts or via social media. Around one-third (n = 12 718) of included participants had prior diagnoses of depression or anxiety and had completed pre-pandemic mental health assessments (between September 2018 and February 2020), allowing prospective investigation of symptom change. RESULTS: Prospective symptom analyses showed small decreases in depression (PHQ-9: -0.43 points) and anxiety [generalised anxiety disorder scale - 7 items (GAD)-7: -0.33 points] and increases in PTSD (PCL-6: 0.22 points). Conversely, retrospective symptom analyses demonstrated significant large increases (PHQ-9: 2.40; GAD-7 = 1.97), with 55% reported worsening mental health since the beginning of the pandemic on a global change rating. Across both prospective and retrospective measures of symptom change, worsening depression, anxiety and PTSD symptoms were associated with prior mental health diagnoses, female gender, young age and unemployed/student status. CONCLUSIONS: We highlight the effect of prior mental health diagnoses on worsening mental health during the pandemic and confirm previously reported sociodemographic risk factors. Discrepancies between prospective and retrospective measures of changes in mental health may be related to recall bias-related underestimation of prior symptom severity.

Microdialysis in cutaneous pharmacology: kinetic analysis of transdermally delivered nicotine.

Direct measurements of cutaneous drug levels and kinetics have long been hampered by lack of appropriate methods. Recently, studies have indicated that microdialysis, a method of continuous in vivo sampling of extracellular fluid, may also be performed in human skin. The present study was designed to evaluate this technique for kinetic analyses of cutaneous drug levels. Using a transdermal nicotine delivery system with 35 mg of nicotine as a model, nicotine levels were determined in the dialysate of human skin by means of high performance liquid chromatography. In vitro studies demonstrated that nicotine levels in the dialysate strictly correlated with nicotine concentrations in the dialyzed medium. In nine healthy male volunteers receiving nicotine by transdermal delivery, nicotine was detectable within 90-180 min, and peak levels of approximately 1000 ng/ml were detected within 240-360 min of patch application. Correlation analyses of the individual data from our subjects revealed that nicotine kinetics were independent of skin barrier function, as assessed by transepidermal water loss, but indicated that the detectable maximum nicotine levels may depend on the location of the probe. In summary, the present study demonstrates that microdialysis may be a novel, powerful tool to study cutaneous pharmacology in vivo.

Direct interaction of antifungal azole-derivatives with calmodulin: a possible mechanism for their therapeutic activity.

Azole derivatives, such as ketoconazole and bifonazole, are well-established antifungal drugs. Recently, these compounds have been reported to have therapeutic efficacy also in inflammatory skin disorders. There is increasing evidence that calmodulin is involved in fungal infections as well as in inflammatory skin diseases. Therefore, we investigated the effects of various antifungal drugs on calmodulin activity, using calmodulin-dependent phosphodiesterase as an indicator for the calmodulin activity. All azole derivatives tested competitively inhibited calmodulin activity with 50% inhibitory concentration values in the low micromolar range. In contrast, antifungal drugs belonging to other chemical classes did not display inhibitory activity. Thus, this study provides evidence that direct interaction with calmodulin might contribute to the therapeutic activity of azole derivatives, particularly to their efficacy in the treatment of inflammatory skin disorders.

Changes of epidermal cell morphology and keratin expression induced by inhibitors of protein kinase C.

Several lines of evidence show protein kinase C as being involved in various regulatory processes in keratinocyte biology, e.g. proliferation and differentiation. In the present study, we investigated the effects of three different inhibitors of protein kinase C, staurosporine, CP 46'665-1, and tiflucarbine, on cell morphology and keratin expression in a non-tumorigenic human keratinocyte cell line (HaCaT cells). Staurosporine, being the most potent inhibitor of protein kinase C activity in vitro, and CP 46'665-1 induced morphological transformation to a fibroblast-like cell shape. In contrast, no changes in cell morphology were observed after exposure to tiflucarbine. The investigation of keratin expression in HaCaT cells grown in the presence of the different compounds revealed the following changes: After 72 h of cultivation, keratins 8 and 18 were still expressed in treated cells, whereas expression of keratin 13 was decreased as compared to control cells. Immunoblotting to detect vimentin demonstrated its absence in treated and control cells. Since tiflucarbine is known as a dual protein kinase C/calmodulin inhibitor whereas staurosporine and CP 46'665-1 do not antagonize calmodulin function, it might be possible that not only protein kinase C but also calmodulin is involved in the process leading to the morphological changes.

Anti-proliferative effects of protein kinase C inhibitors in human keratinocytes.

Various lines of evidence indicate that protein kinase C, a key enzyme in transmembraneous signal transduction, is involved in the regulation of keratinocyte proliferation. In the present study we have investigated the effects of various structurally unrelated protein kinase C inhibitors on the proliferation of HaCa T cells, a non-tumorigenic human keratinocyte cell line. All protein kinase C inhibitors dose-dependently inhibited cell proliferation as assessed by the incorporation of radioactively labelled thymidine and amino acids as well as the increase in total protein content in keratinocytes. The potencies of the drugs to inhibit cell proliferation were strongly correlated to their inhibitory potency on purified protein kinase C, displaying a correlation coefficient of 0.97. Methotrexate, an anti-proliferative drug, was found not to inhibit protein kinase C. Therefore, our data provide evidence that protein kinase C is crucially involved in the regulation of keratinocyte proliferation but is not the only target of anti-proliferative drug action.

Polymyxin B is a selective and potent antagonist of calmodulin.

Polymyxin B, a cyclic peptide antibiotic, is considered to be a rather selective antagonist of protein kinase C. This drug is therefore widely used to evaluate the involvement of protein kinase C in cellular processes. In the present study, we investigated the effects of polymyxin B on the activity of calmodulin-dependent cyclic 3':5'-nucleotide phosphodiesterase in vitro. The drug potently inhibited this enzyme (IC50 80 nM in the presence of 500 microM Ca2+), while about 200-fold higher concentrations were required to inhibit protein kinase C to the same extent. Phosphodiesterase inhibition was competitive with respect to Ca2+ and calmodulin. Evidence for the formation of a complex between polymyxin B and calmodulin was obtained by polyacrylamide gel electrophoresis under non-denaturing conditions, and by affinity chromatography of calmodulin on polymyxin B-agarose. We therefore suggest that, at least in vitro, polymyxin B is a potent and selective inhibitor of calmodulin.

Inhibition of a model of in vitro granuloma formation by tetracyclines and ciprofloxacin. Involvement of protein kinase C.

BACKGROUND AND DESIGN: Granulomatous inflammation is a common component of many diseases. In this study the ability of commonly used antibiotics to inhibit an in vitro model of granuloma formation were studied. The effect of protein kinase C inhibition in this system was also investigated. RESULTS: Ampicillin, cephalothin, metronidazole, rifampin, isoniazide, erythromycin, and clindamycin were inactive in inhibiting granuloma formation. Tetracycline, doxycycline, minocycline, and ciprofloxacin produced dose-dependent inhibition of the granuloma model in concentrations between 10(-4) and 10(-6) mol/L. The approximate order of descending potency was doxycycline equals minocycline greater than tetracycline greater than ciprofloxacin. The same drugs were tested for the ability to inhibit protein kinase C. Drugs inactive in the granuloma model had no effect on protein kinase C activity. The tetracyclines and ciprofloxacin all caused a dose-dependent inhibition of protein kinase C activity in the same order of relative potency as was found for inhibition of granuloma formation. CONCLUSIONS: These data demonstrate a previously unappreciated activity of the tetracyclines and ciprofloxacin. Inhibition of granuloma formation helps to account for the activity of these drugs in the severest forms of inflammatory acne.

Effect of protein kinase inhibitors on ACTH-stimulated aldosterone production in rat zona glomerulosa cells.

In order to obtain further evidence for the involvement of protein kinases in the short-term ACTH-stimulated aldosterone synthesis in rat zona glomerulosa cells, the effects of three different compounds with protein kinase inhibitory properties were investigated. Staurosporine, H-7 and trifluoperazine inhibited ACTH-stimulated aldosterone release in a dose-dependent manner. While the inhibitory effect of H-7 was reversible upon washing of the cells with inhibitor-free medium, the inhibition was maintained in cells treated with staurosporine or trifluoperazine. In contrast to the stimulated production, basal release of aldosterone even at the highest drug concentrations tested was not completely inhibited. We thus conclude that protein kinases may play a crucial role in short-term ACTH-stimulated aldosterone production in rat glomerulosa cells.

The involvement of protein kinase C in proliferation and differentiation of human keratinocytes--an investigation using inhibitors of protein kinase C.

Protein kinase C, the major cellular receptor for tumour-promoting phorbol esters, has been suggested as playing a key role in the regulation of proliferation and differentiation of epidermal cells. In the present study, we investigated the effects of various well-characterized inhibitors of protein kinase C on proliferation and differentiation of SV 40-transformed and normal human keratinocytes. The drugs were found to inhibit cell proliferation in a dose-dependent manner, displaying similar effects in both cell types and reflecting their potencies in inhibiting purified protein kinase C. In contrast, keratinocyte differentiation induced by treatment with a calcium ionophore or spontaneously, i.e. by exposure of cells grown in the presence of low calcium concentration (0.06 mM) to normal calcium concentration (1.6 mM), was not inhibited by the compounds tested. The potent protein kinase C inhibitor, staurosporine, was found even to enhance cell differentiation. Therefore, the present study provides evidence that the classical protein kinase C pathway plays a critical role in the regulation of keratinocyte proliferation rather than in calcium-induced differentiation.

The antipsoriatic drug, anthralin, inhibits protein kinase C--implications for its mechanism of action.

In psoriatic patients, anthralin is known to attenuate lesional inflammation, but often generates perilesional dermatitis. This phenomenon is well reflected by the contrasting action of anthralin on human leukocytes. The release of reactive oxygen species (ROS) is inhibited by anthralin in phorbol ester-activated leukocytes, whereas anthralin directly induces this cellular response in unstimulated cells. In order to elaborate further the underlying mechanisms, we compared the kinetics of anthralin and different well-characterized stimuli, including the phorbol ester, phorbol-12-myristate-13-acetate, in this test system. Compared with standard stimuli, anthralin only marginally induced the release of ROS from human leukocytes and displayed different kinetics. Protein kinase C (PKC), the major cellular phorbol ester receptor, is considered to be involved in the regulation of this cellular response. Furthermore, its involvement in the pathophysiology of psoriasis has been suggested. Therefore, we also investigated the effects of anthralin on purified PKC. Anthralin was found to inhibit the enzyme activity in a dose-dependent manner but not to display any stimulatory effects. The present results provide first evidence that the therapeutic activity of anthralin, at least in part, might be mediated by inhibition of PKC.

Effects of tiflucarbine as a dual protein kinase C/calmodulin antagonist on proliferation of human keratinocytes and release of reactive oxygen species from human leukocytes.

Various studies have suggested that calmodulin (CaM) is involved in the pathophysiology of psoriasis. Protein kinase C (PKC) is also accepted as playing a regulatory role in cell proliferation as well as in inflammatory processes. Therefore, we investigated the effects of the known CaM antagonist tiflucarbine (BAY/TVX P 4495) on two cellular systems related to the major clinical symptoms of psoriasis: proliferation of cultured human keratinocytes (HaCa T cell line) and release of reactive oxygen species (ROS) from human polymorphonuclear leukocytes (PMNL). Tiflucarbine inhibited both cellular responses in a dose dependent manner. Furthermore, tiflucarbine directly affected PKC, and may thus be considered to be a dual PKC/CaM antagonist with putative antipsoriatic activity. The effects of tiflucarbine on the different parameters were compared with those of the structurally unrelated dual PKC/CaM inhibitor W-7 and those of the potent PKC inhibitor staurosporine. The potencies of all three compounds were found to be in the same range as their PKC-inhibiting potency. Our data indicate that PKC, rather than CaM, may play a regulatory role in the release of ROS as well as in keratinocyte proliferation. Therefore, inhibition of PKC in general might have a therapeutic benefit in psoriasis.

Anthralin: how does it act and are there more favourable derivatives?

Anthralin is still the most effective and safest therapeutic agent for treatment of psoriasis. Our data may assist toward an understanding of its mode of action and introduce new derivatives, more antiproliferative and less toxic than anthralin in vitro. Anthralin exerts a direct effect on keratinocytes and leukocytes. In time-lapse studies it significantly prolonged the prophase of mitotic keratinocytes in subtoxic doses and suppressed the expression of keratin 6 mRNA in the immediately suprabasal layer of psoriatic epidermis in vivo. Anthralin inhibits the transformation of lymphocytes and the release of reactive oxygen species from activated leukocytes, in vitro. We provide evidence that these effects of anthralin are mediated by protein kinase C. Twelve new hydrophilic derivatives of anthralin, including a 1,8-dimethoxy compound, as well as C-2 and C-10 substituted anthrones were tested on human keratinocytes. The antiproliferative effect of those derivatives bearing lacton rings at a C-10, consisting of 4, 5, or 6 C atoms, exceeded that of anthralin and were equally or less cytotoxic than the parent drug. These compounds had no pro-drug character in vitro, since they did not metabolize via anthralin, as shown by HPLC. These data indicate that there may be anthralin derivatives with more favourable properties for topical therapy than anthralin itself.

[Simplification of the production of plastination histologic preparations through the use of a grinding machine]. / Vereinfachung der Herstellung plastinationshistologischer Präparate durch Einsatz einer Schleifmaschine.

A new histological method basing on the plastination technique (v. Hagens, Tiedemann, Kriz 1987) has recently been developed and applied to research in human fetal development. This plastination histology shows some evident advantages compared with the classical histological techniques. As the application of this new method has been very time consuming up to now, only some special questions of basic research have been investigated. We here present the use of a grinding-machine, which can be used to partially mechanize the procedure of plastination histology.

[Development of the ligamentum capitis femoris and the artery with the same name]. / Entwicklung des Ligamentum capitis femoris und der gleichnamigen Arterie.

A morphological study concerning the development of the ligamentum teres and its artery was performed by investigating 300-600 microns thick epoxy resin impregnated sections through the hip joints of human fetuses and newborn children. In addition, the hip joints were macroscopically studied in three fetuses and in two newborn children. After photographical documentation the ligamenta teretes of these hips were processed into paraffin section. Within the ligamentum teres areas of different structures can clearly be distinguished during fetal development. The subsynovial layer consists of dense connective tissue containing numerous large cells. The major central portion of the ligament is formed by dense regular connective tissue. The blood vessels of the ligament are always surrounded by a layer of loose connective tissue. Adipose tissue firstly starts to spread in the postnatal period mainly occurring around the vessels. In the newborn child dense connective tissue is most abundant within the ligamentum teres. Thus it is considered to be especially strong. The femoral capitis artery is already present in 9 week-old fetuses. In 13 week-old fetuses branches of the femoral capitis artery enter the femoral head through cartilaginous channels and continuously maintain within the femoral head during further development. With the growing age of the fetuses the number of branches of the femoral capitis artery increases. Anastomoses between the cartilaginous channels of the femoral capitis artery and the medial and lateral circumflexal femoral artery can be found neither prenatally nor postnatally.

Ultrastructural changes in onychomycosis during the treatment with bifonazole/urea ointment.

In the present study, we investigated the effects of bifonazole/urea ointment, a novel topical drug for the treatment of onychomycosis, on the ultrastructure of normal and fungus-infected human toenails by scanning electron microscopy. Nails were treated with the drug ex vivo and compared to untreated controls. During treatment, the corneocyte layers disintegrated, and the structure markedly loosened. After 5 days of treatment, the morphology of fungal cells, found at the undersurface of infected nails, was changed due to the antifungal drug action. The present data demonstrate that by the use of this drug, the agent effectively penetrates the nail plate and affects the fungal cells.

Selective calmodulin antagonists fail to inhibit phorbol ester-induced superoxide anion release from human neutrophils: effects of antifungal azole derivatives.

The ability of antifungal azole derivatives to inhibit superoxide anion release from human leucocytes and the relevance of their documented calmodulin (CaM) antagonism was investigated with respect to anti-inflammatory drug activity. Econazole, miconazole and clotrimazole were found to inhibit phorbol ester-induced release of superoxide anions from human polymorphonuclear leucocytes effectively with IC50 values in the range of 36-162 mumol/l. In contrast, bifonazole and ketoconazole produced minimal or no inhibition, thus suggesting that mechanisms other than inhibition of superoxide anion release may largely account for their clinical activity in inflammatory skin disorders. The selective CaM antagonist J-8, which was used as a reference, failed to inhibit the release process, whereas W-7 as a dual CaM/protein kinase C inhibitor induced dose-dependent inhibition. When tested on protein kinase C activity in vitro, econazole, miconazole and clotrimazole were inhibitory, but bifonazole and ketoconazole were without significant effect. It is thus concluded that inhibition of superoxide anion release reflects the ability of these drugs to inhibit protein kinase C, but not their potency to antagonize CaM. Given the role of reactive oxygen species in tissue damage by neutrophils, we propose protein kinase C, rather than CaM, as another potential target of anti-inflammatory therapy.

Potent antagonism of calmodulin activity in vitro, but lack of antiproliferative effects on keratinocytes by the novel leukotriene biosynthesis inhibitor MK-886.

MK-886, a leukotriene biosynthesis inhibitor, which prevents the translocation and activation of 5-lipoxygenase, has been proposed as an effective drug for the treatment of inflammatory disorders, including psoriasis. In the present study, we investigated the effects of MK-886 on calmodulin as a potential target protein of anti-inflammatory drug activity, and on the proliferation of cultured human keratinocytes, a calmodulin-dependent cellular response with indicative value for antipsoriatic drug activity. Despite potent calmodulin-antagonistic activity in vitro, MK-886 failed to block cell proliferation in a human keratinocyte line, whereas trifluoperazine, a well characterized calmodulin antagonist with similar effects on calmodulin activity in our in vitro assays, inhibited cell proliferation in a dose-dependent manner. Further investigations on the mechanism of action revealed that, in contrast with trifluoperazine, calmodulin antagonism by MK-886 in vitro is likely to be mediated at the level of the allosteric calmodulin-recognition site of phosphodiesterase, rather than by binding to calmodulin itself. Therefore, our data do not conflict with the proposed role of calmodulin in the regulation of cell proliferation, but demonstrate that drug-induced antagonism of calmodulin, detected by inhibition of calmodulin-dependent enzymes in vitro, is not necessarily linked to antiproliferative activity in human keratinocytes.

Anti-inflammatory actions of benzoyl peroxide: effects on the generation of reactive oxygen species by leucocytes and the activity of protein kinase C and calmodulin.

For many years, benzoyl peroxide has been used as a topical treatment for acne. Although the drug has been shown to interfere with a variety of pathways, believed to be of importance in the aetiopathogenesis of acne, its mechanism of action is thought to be principally antibacterial. Recent circumstantial evidence suggests that protein kinase C might serve as an additional pharmacological target of benzoyl peroxide. In the present study, we investigated the effects of benzoyl peroxide on the release of reactive oxygen species, regulated by protein kinase C and calmodulin, from human neutrophils, a potentially important step in acne inflammation. Micromolar drug concentrations were found to inhibit the release of reactive oxygen species, but there was marked drug-induced cytotoxicity in neutrophils. However, when tested in cell-free assays, benzoyl peroxide displayed marginal inhibition of protein kinase C, but failed to antagonize calmodulin. Further investigations on its mechanism of action revealed non-specific interference with nucleotide binding sites. Therefore, the data presented here indicate that, in contrast with our previous findings with tetracycline derivatives, the clinical anti-inflammatory activity of benzoyl peroxide is unlikely to be mediated by protein kinase C or calmodulin. The differential interaction of drugs with protein kinase C and calmodulin might help to explain their different clinical usefulness in various degrees of acne severity.