New insights into smoking and urinary tract infections during pregnancy using pregnancy-pair design: A population-based register study.

INTRODUCTION: Pregnancy itself predisposes to urinary tract infections (UTI). There appears to be a higher prevalence of infections and genitourinary diseases among pregnant smokers than among non-smokers. The present study is a retrospective observational register study aiming to investigate whether maternal smoking is associated with the prevalence of UTIs during pregnancy by utilizing a pregnancy-pair analysis. MATERIAL AND METHODS: Information about pregnancies and maternal smoking was obtained from the Finnish Medical Birth Register. The study sample consisted of all singleton pregnancies (n = 723 433) of women giving birth between January 2006 and December 2018 in Finland. Information on maternal smoking was collected in three categories: (1) non-smoking; (2) quit smoking during the first trimester; and (3) continued smoking throughout the pregnancy. Information about maternal UTI diagnoses during pregnancy was received from the Hospital Discharge Register and the Medical Birth Register. UTIs were categorized as lower and upper UTIs according to the International Statistical Classification of Diseases and Related Health Problems (ICD)-10 diagnosis codes. Risks were calculated as odds ratios (OR) by logistic regression with 95% confidence intervals (CI) further adjusted for maternal characteristics (aOR). Finally, pregnancy-pair analyses were performed: mothers who had changed smoking status (no smoking/any smoking) between consecutive pregnancies (n = 27 246 pregnancy-pairs) were analyzed as one cluster and compared with non-smokers. RESULTS: Smokers had UTIs more often compared with the non-smokers. The association was even stronger among those who continued to smoke (aOR 1.60, 95% CI 1.51-1.70) than among those who smoked only during the first trimester (aOR 1.27, 95% CI 1.18-1.37) compared with non-smokers. In pregnancy-pair analysis, smoking was associated with upper UTIs during pregnancy (OR 1.49, 95% CI 1.05-2.12) compared with non-smokers, but after the adjustments this association was attenuated (aOR 1.27, 95% CI 0.88-1.82). No association in lower UTIs was observed in the pregnancy-pair design. CONCLUSIONS: Maternal smoking was associated with a higher prevalence of UTIs during pregnancy in the standard comparison. The observed association was fully attenuated in the pregnancy-pair analysis, in which smoking was dichotomized. This study suggests that the association between maternal smoking during pregnancy and adverse maternal health effects might be more complex than previously thought.

Design of a prospective follow-up study on early parenthood and smoking behaviour during pregnancy in Finnish primary healthcare.

Aims: The primary aim of the study is to explore different factors affecting parents' smoking behaviour, and especially how smoking may be connected with individual differences in the psychological process of becoming a parent. In the current paper, we present the study design together with basic information on the study population. Methods: The Central Satakunta Maternity and Child Health Clinic (KESALATU) Study is an ongoing prospective follow-up study in primary healthcare of the Satakunta region of southwest Finland. Families were recruited during their first maternity clinic visit between 1 September 2016 and 31 December 2019, and participation will continue until the child is 1.5 years of age. The study combines different sources and types of data: e.g. routine data obtained from primary healthcare clinic records, specific parental self-report data and data from a new exhaled carbon monoxide meter indicating maternal smoking. The data are collected using frequently repeated assessments both during pregnancy and postnatally. The methods cover the following areas of interest: family background factors (including smoking and alcohol use), self-reported parental-foetal/infant attachment and mentalization, self-reported stress, depression and quality of life. Results: 589 pregnant women and their partners were asked to participate in the study during the collection time period. The final study population consisted of 248 (42.1%) pregnant women and 160 (27.1%) partners. Conclusions: The new methods and study design have the potential to increase our understanding about the link between early parenting psychology, prenatal psychosocial risk factors and parental health behaviour.

Externally added cystatin C reduces growth of A375 melanoma cells by increasing cell cycle time.

Some secreted cysteine protease inhibitors of the cystatin family appear to affect intracellular proteolysis and growth of human cells, as a result of internalization. Here, we studied the effects of external addition of the most abundant human cystatin, cystatin C, on viability and proliferation of cancer cells in culture. A dose-dependent decrease in viable cells was seen for A375 melanoma, MCF-7 breast cancer, and PC-3 prostate cancer cells cultured in 1-5 µm cystatin C after 24 h. Real-time assessment of growth rates in A375 cell cultures for 48 h by digital holographic microscopy showed an increased doubling time for cells cultured in the presence of 5 µm cystatin C (20.1 h) compared with control cells (14.7 h). A prolonged doubling time was already observed during the first 12 h, indicating a rapid general decrease in cell proliferation at the population level. Tracking of individual cells in phase holographic images showed that dividing cells incubated with 5 µm cystatin C underwent fewer mitoses during 48 h than control cells. In addition, the time between cell divisions was longer, especially for the first cell cycle. Incubation with the variant W106F-cystatin C (with high cellular uptake rate) resulted in a lower number of viable cells and a prolonged doubling time than when cells were incubated with wild-type cystatin C, but no effect was observed for (R24A,R25A)-cystatin C (low cellular uptake). Thus, cystatin C causes prolonged cell division leading to decreased proliferation of melanoma cells, and internalization seems to be a prerequisite for this effect.

Secreted cystatins decrease proliferation and enhance apoptosis of human leukemic cells.

Cysteine proteases are implicated in proteolysis events favoring cancer cell growth, spread, and death by apoptosis. Herein, we have studied whether the net growth and survival of the leukemic cell lines Jurkat, U937, and HL-60 are affected by external addition of five proteins acting as natural cysteine protease inhibitors. None of the cystatins examined (A, C, D, and E/M) or chagasin showed consistent effects on Fas-induced apoptosis when evaluated at 1 µm. In contrast, when the intrinsic apoptosis pathway was activated by hydrogen peroxide, addition of cystatin D augmented caspase-3-like activity within all three cell lines. Flow cytometric analysis of U937 cells also showed increased numbers of annexin V-positive cells when hydrogen peroxide was used to initiate apoptosis and cells were cultured in the presence of cystatin D or C. Moreover, stimulation of hydrogen peroxide-induced apoptotic U937 cells with either cystatin C or D resulted in a dose-dependent decrease in the number of cells. Cell viability was also decreased when U937 cells were cultured in the presence of cystatin C or D (1-9 µm) only, demonstrating that these cystatins can reduce cell proliferation by themselves in addition to enhancing apoptosis induced by oxidative stress. These effects on U937 cells were paralleled by internalization of cystatins C and D, indicating these effects are caused by downregulation of intracellular proteolysis. External addition of cystatins C and D to HL-60 and Jurkat cells demonstrated similar degrees of cystatin D uptake and decreased viability as for U937 cells, indicating that these effects are general for leukemic cells.

Self-Efficacy, Psychological Flexibility, and Basic Needs Satisfaction Make a Difference: Recently Graduated Psychologists at Increased or Decreased Risk for Future Health Issues.

The transition from university to working life appears a critical period impacting human service workers' long-term health. More research is needed on how psychological factors affect the risk. We aimed to investigate how subgroups, based on self-efficacy, psychological flexibility, and basic psychological needs satisfaction ratings, differed on self-rated health, wellbeing, and intention to leave. A postal survey was sent to 1,077 recently graduated psychologists in Sweden (≤3 years from graduation), response rate 57.5%, and final sample 532 (75% women and 23% men). A hierarchical cluster analysis resulted in a satisfactory eight-cluster solution. We identified two at-risk subgroups, displaying the lowest scores on health and wellbeing, and one potential low-risk subgroup with the highest ratings on said variables. The "Low risk?" group rated high on all three psychological constructs, a positive transition to working life, a work environment where resources balanced relatively high emotional demands, good health, and wellbeing. Almost the complete opposite ratings characterized the potential risk groups. "Quitting?" scored significantly higher than "Getting sick?" on self-efficacy and psychological flexibility as well as actively seeking new employment and reporting daily thoughts on leaving the profession. We suggest that a combination of low self-efficacy and psychological flexibility could increase the risk of individuals staying despite suboptimal working conditions. With combined higher self-efficacy and psychological flexibility, individuals in similar circumstances appear more inclined to quit. We conclude that the ways recently graduated psychologists rate their self-efficacy, psychological flexibility, and basic needs satisfaction appear to be reflected in their self-rated health and wellbeing.

Maternal Smoking and Hospital Treatment During Pregnancy.

INTRODUCTION: Previous research suggests that young maternal age, smoking, hospitalization during a previous pregnancy, and poor self-rated health could be risk factors for prenatal hospitalization. METHODS: The objective of this retrospective observational register study was to investigate if maternal smoking during pregnancy is associated with mother's need for hospital treatment during pregnancy. The study population consists of all singleton pregnancies (n = 961 127) in 1999-2015 in Finland. Information on maternal smoking was received from the Medical Birth Register in three classes: nonsmoker, quit smoking in the first trimester, and continued smoking throughout the pregnancy. These data were linked with the Hospital Discharge Register data and analyzed according to ICD-10 chapters. RESULTS: 10.7% of women continued to smoke after the first trimester. After adjusting for confounding factors women in both smoking groups had more hospital treatment compared with nonsmokers. Especially outpatient treatment was more common among mothers who continued to smoke compared to those who quit smoking in the first trimester in several ICD-10 chapters. Compared to non-smokers, aOR for mental and behavioral disorders (F00-F99) was 2.14 (95% confidence interval 2.00-2.30) in the quit smoking group and 3.88 (3.71-4.06) in the continued smoking group. Similarly, aOR for respiratory diseases (J00-J99) was 1.26 (1.15-1.39) and 1.61 (1.52-1.71), respectively and aOR for genitourinary diseases (N00-N99) was 1.10 (1.03-1.17) and 1.29 (1.23-1.35), respectively. Some similar findings were made also in inpatient care. Some similar findings were made also in inpatient care. CONCLUSIONS: Women who smoke during pregnancy seem to require more hospital care for various reasons. These findings emphasize the importance of actions for smoking cessation during pregnancy and women should be encouraged to quit as early as possible. IMPLICATIONS: Maternal smoking during pregnancy is associated with greater rates of both outpatient and inpatient hospital care during pregnancy. Women who quit smoking had a similar risk for hospital care during pregnancy with nonsmokers in certain diagnosis chapters, which is very motivational and could be used as an informational tool in prenatal clinics to encourage smoking cessation as it is never too late to quit smoking during pregnancy.

Low-level internalization of cystatin E/M affects legumain activity and migration of melanoma cells.

The ratio between proteases and their inhibitors is unbalanced in cancer. The cysteine protease inhibitor cystatin C is internalized by some cancer cells, which affects cellular properties. Here we aimed to investigate if uptake of cystatin C and the related inhibitor cystatin E/M occur in melanoma cell lines and to evaluate to what extent the uptake affects the legumain activity that is typically increased in melanoma. First we studied the basic expression, secretion, and intracellular content of all type 2 cystatins as well as expression and activity of their possible target enzymes legumain and cathepsin B in MDA-MB-435S, A375, and C8161 melanoma cells. Legumain activity was measureable in all cell lines, and of the potential legumain inhibitors, cystatin C, E/M, and F, cystatin C was the one mainly produced. All cells internalized cystatin C added to culture media, leading to increased intracellular cystatin C levels by 120-200%. Cystatin E/M was internalized as well but at a modest rate. The effects on intracellular legumain activity were nevertheless pronounced, probably because the cells lacked this inhibitor, and its affinity for legumain is 100-fold higher than that of cystatin C. Likewise, the low-degree uptake resulted in reduced migration and invasion of A375 cells in Matrigel to an extent comparable with the W106F variant of cystatin C with optimal uptake properties and resulting in much higher intracellular levels. Thus, cystatin E/M appears to be a good candidate to efficiently down-regulate the increased legumain activity, possibly important for the malignant phenotype of melanoma cells.

Cystatin C properties crucial for uptake and inhibition of intracellular target enzymes.

To elucidate the molecular requirements for cancer cell internalization of the extracellular cysteine protease inhibitor cystatin C, 12 variants of the protein were produced and used for uptake experiments in MCF-7 cells. Variants with alterations in the cysteine cathepsin binding region ((Δ1-10)-, K5A-, R8G-, (R8G,L9G,V10G)-, (R8G,L9G,V10G,W106G)-, and W106G-cystatin C) were internalized to a very low extent compared with the wild-type inhibitor. Substitutions of N39 in the legumain binding region (N39K- and N39A-cystatin C) decreased the internalization and (R24A,R25A)-cystatin C, with substitutions of charged residues not involved in enzyme inhibition, was not taken up at all. Two variants, W106F- and K75A-cystatin C, showed that the internalization can be positively affected by engineering of the cystatin molecule. Microscopy revealed vesicular co-localization of internalized cystatin C with the lysosomal marker proteins cathepsin D and legumain. Activities of both cysteine cathepsins and legumain, possible target enzymes associated with cancer cell invasion and metastasis, were down-regulated in cell homogenates following cystatin C uptake. A positive effect on regulation of intracellular enzyme activity by a cystatin variant selected from uptake properties was illustrated by incubating cells with W106F-cystatin C. This resulted in more efficient down-regulation of intracellular legumain activity than when cells were incubated with wild-type cystatin C. Uptake experiments in prostate cancer cells corroborated that the cystatin C internalization is generally relevant and confirmed an increased uptake of W106F-cystatin C, in PC3 cells. Thus, intracellular cysteine proteases involved in cancer-promoting processes might be controled by cystatin uptake.

Cystatins--Extra- and intracellular cysteine protease inhibitors: High-level secretion and uptake of cystatin C in human neuroblastoma cells.

Cystatins are present in mammals, birds, fish, insects, plants, fungi and protozoa and constitute a large protein family, with most members sharing a cysteine protease inhibitory function. In humans 12 functional cystatins exist, forming three groups based on molecular organisation and distribution in the organism. The type 1 cystatins (A and B) are known as intracellular, type 2 cystatins (C, D, E/M, F, G, S, SN and SA) extracellular and type 3 cystatins (L- and H-kininogen) intravascular proteins. The present paper is focused on the human cystatins and especially those of type 2, which are directed (with signal peptides) for cellular export following translation. Results indicating existence of systems for significant internalisation of type 2 cystatins from the extracellular to intracellular compartments are reviewed. Data showing that human neuroblastoma cell lines generally secrete high levels, but also contain high amounts of cystatin C are presented. Culturing of these cells in medium containing cystatin C at concentrations found in body fluids resulted in increased intracellular cystatin C, as a result of an uptake process. At immunofluorescence cytochemistry a pronounced vesicular cystatin C staining was observed. The simplistic denotation of the type 2 cystatins as extracellular inhibitors is thus challenged, and possible biological functions of the internalised cystatins are discussed. To illustrate the special case of high cellular cystatin content seen in cells of patients with hereditary cystatin C amyloid angiopathy, expression vectors for wild-type and L68Q mutated cystatin C were used to transfect SK-N-BE(2) cells. Clones overexpressing the two variants showed increased secreted levels of cystatin C. Within the cells the L68Q variant appeared to mainly localise to the endoplasmic reticulum rather than to acidic vesicular organelles, indicating limitations in the transport out from the cell rather than increased uptake as explanation for the elevated cellular cystatin levels seen in hereditary cystatin C amyloid angiopathy.

Macrophage responses to interferon-gamma are dependent on cystatin C levels.

The aim of the present investigation was to elucidate possible effects of cystatin C on inflammatory responses mediated by macrophages. Previously it has been shown that in vitro treatment of murine peritoneal macrophages with interferon-gamma (IFN-gamma) causes a down-regulation of cystatin C secretion. To investigate whether such changes in cystatin C expression in turn can affect inflammatory responses mediated by macrophages, we have compared effects of IFN-gamma on macrophages isolated from wild-type (cysC(+/+)) and cystatin C knockout (cysC(-/-)) mice. It was shown that IFN-gamma-primed cysC(-/-) macrophages exhibit significantly higher interleukin-10 (IL-10) but lower tumor necrosis factor-alpha (TNF-alpha) expression, and reduced nuclear factor (NF)-kappaB p65 activation, compared to similarly primed cysC(+/+) cells. Exogenously added cystatin C enhanced IFN-gamma-induced activation of NF-kappaB p65 and increased mRNA levels for inducible NO synthase (iNOS) in cysC(-/-) macrophages as well as levels of nitric oxide and TNF-alpha in the cell culture medium, in agreement with an enhanced pro-inflammatory response. Accordingly, IFN-gamma-induced IL-10 mRNA expression in cysC(-/-) macrophages was down-regulated by exogenously added cystatin C. Taken together, our data provide evidence that changes in cystatin C levels alter macrophage responses to IFN-gamma. The latter down-regulates the production of cystatin C, which leads to a suppressed inflammatory condition with enhanced IL-10 levels and down-regulated TNF-alpha and NF-kappaB. It is concluded that cystatin C through this effect can act as an immunomodulatory molecule.

Internalization of cystatin C in human cell lines.

Altered protease activity is considered important for tumour invasion and metastasis, processes in which the cysteine proteases cathepsin B and L are involved. Their natural inhibitor cystatin C is a secreted protein, suggesting that it functions to control extracellular protease activity. Because cystatins added to cell cultures can inhibit polio, herpes simplex and coronavirus replication, which are intracellular processes, the internalization and intracellular regulation of cysteine proteases by cystatin C should be considered. The extension, mechanism and biological importance of this hypothetical process are unknown. We investigated whether internalization of cystatin C occurs in a set of human cell lines. Demonstrated by flow cytometry and confocal microscopy, A-431, MCF-7, MDA-MB-453, MDA-MB-468 and Capan-1 cells internalized fluorophore-conjugated cystatin C when exposed to physiological concentrations (1 microm). During cystatin C incubation, intracellular cystatin C increased after 5 min and accumulated for at least 6 h, reaching four to six times the baseline level. Western blotting showed that the internalized inhibitor was not degraded. It was functionally intact and extracts of cells exposed to cystatin C showed a higher capacity to inhibit papain and cathepsin B than control cells (decrease in enzyme activity of 34% and 37%, respectively). The uptake of labelled cystatin C was inhibited by unlabelled inhibitor, suggesting a specific pathway for the internalization. We conclude that the cysteine protease inhibitor cystatin C is internalized in significant quantities in various cancer cell lines. This is a potentially important physiological phenomenon not previously described for this group of inhibitors.

Cystatin C is highly expressed in the human male reproductive system.

Cystatin C displays the strongest inhibitory activity of all cystatins toward lysosomal cysteine proteases in general and has a widespread distribution in human tissues and body fluids, including seminal plasma. The aim of this study was to investigate the distribution of cystatin C in the male reproductive system. Immunohistochemistry revealed a widespread distribution of cystatin C in normal tissues from the testis, epididymis, vas deferens, seminal vesicle, and prostate gland. Immunoreactive cystatin C was localized in basal and secretory epithelial cells, but also in neuroendocrine cells in the prostate, identified by immunostaining for chromogranin A. On adjacent tissue sections, we demonstrated local production of cystatin C utilizing nonradioactive in situ hybridization with a 201-base-long digoxigenin-labeled antisense RNA probe specific for the cystatin C transcript. Staining patterns obtained by immunohistochemistry and in situ hybridization correlated well. Enzyme-linked immunosorbent assay for quantitative analysis of cystatin C demonstrated that cystatin C was present at high concentrations in tissue homogenates from all locations investigated, compared to liver, muscle, spleen, and other general tissues. Western blotting of tissue homogenates revealed a predominant 15-kd cystatin C immunoreactive component in accordance with previous findings in other organs. Quantitative real-time polymerase chain reaction analysis to determine messenger RNA levels in whole tissue extracts showed that the cystatin C gene is highly expressed in the seminal vesicles and the prostate gland, indicating that the major amount of cystatin C in the male reproductive organs and seminal plasma is produced by cells in these 2 tissues. It is concluded that cystatin C is highly expressed and widely distributed throughout the male genital tract, suggesting that cystatin C is an important regulator for normal and pathological proteolysis in the male reproductive system.

Cathepsin B in the rat eye.

BACKGROUND: Cathepsin B is a mammalian cysteine protease. The enzyme has been suggested to participate in the patophysiological processes of keratoconus as well as in the corneal response to infectious agents. This study describes the localization of cathepsin B in the rat eye. METHODS: Cathepsin B was identified in rat ocular tissues by Western blotting and immunohistochemistry. Cathepsin B mRNA levels were analyzed in the tissues by quantitative real-time cDNA amplification (QRT-PCR). RESULTS: Cathepsin B is present in the epithelium, in stromal cells and in the endothelium of the cornea. It is also present in the epithelium lining the ciliary processes, in occasional stromal cells in the iris, in the anterior subcapsular lens epithelium and in various cell types in the retina. At all locations cathepsin B is present in cytoplasmic granules, presumably lysosomes. QRT-PCR analysis detected cathepsin B mRNA in all these tissues in amounts correlating to the immunodetection results, suggesting that the enzyme detected is locally produced. CONCLUSIONS: Cathepsin B is present in several tissues and cell types throughout the rat eye. It is localized to cytoplasmic granules, presumably lysosomes. Our results suggest that it is probably also produced in the same cell types.

Regulated expression and intracellular localization of cystatin F in human U937 cells.

Cystatin F is a cysteine peptidase inhibitor recently discovered in haematopoietic cells by cDNA cloning. To further investigate the expression, distribution and properties of the native human inhibitor the promyeloid cell line U937 has been studied. The cells expressed relatively large quantities of cystatin F, which was found both secreted and intracellularly. The intracellular levels were unusually high for a secreted cystatin ( approximately 25% of the cystatin F in 2- or 4-day culture medium). By contrast, U937 cells contained only 3-4% of the related inhibitor, cystatin C. Cystatin F purified from lysates of U937 cells showed three major forms carrying two, one or no carbohydrate chains. Immunocytochemistry demonstrated a marked cytoplasmic cystatin F staining in a granular pattern. Double staining with a marker for endoplasmic reticulum revealed no colocalization for cystatin F. Analysis of the promoter region of the cystatin F gene (CST7) showed that it, like that of the cystatin C gene (CST3), is devoid of typical TATA- and CAAT-box elements. In contrast to the cystatin C promoter, it does not contain multiple Sp1 binding sites, but has a unique site for C/EBPalpha, possibly explaining the restricted expression of the cystatin F gene. Cells stimulated with all-trans retinoic acid to differentiate them towards a granulocytic pathway, showed a strong ( approximately 18-fold) down-regulation of intracellular cystatin F and almost abolished secreted levels of the inhibitor. Stimulation with tetradecanoyl phorbol acetate, causing monocytic differentiation, also resulted in down-regulation (two fold to threefold) of cystatin F expression, whereas the cystatin C expression was essentially unaltered in both experiments. The results suggest that cystatin F as an intracellular cysteine peptidase inhibitor with readily regulated expression, may be a candidate to control the cysteine peptidase activity known to be essential for antigen presentation in different blood cell lineages.