Side populations from cervical-cancer-derived cell lines have stem-cell-like properties.

The target cells for the transforming mutations caused by high-risk human papillomavirus (HPV) infection could be the stem cells of the uterine cervical epithelium, generating particular cancer stem cells (CSCs). The aim of this study was to identify and characterize the CSCs from cervical-cancer-derived cell lines. The ability of SiHa, CaLo, and C-33A cell lines to efflux Hoechst 33342 was evaluated by flow cytometry and cells from the corresponding side populations (SPs) and nonside populations (NSPs) were analyzed for their cell-cycle status (pyronin Y) and their mRNA levels of ABC transporter family members (with qPCR). Specific markers (α6-integrin(bri)/CD71(dim), CK17) of normal epithelial stem cells were evaluated by flow cytometry. The biological properties of these cells were analyzed, including their colony heterogeneity, repopulation, and anchorage-independent colony formation. We identified SPs (around 3 %) in the SiHa and CaLo cell lines, more than 70 % of which were in G0 phase and strongly expressed ABC transporters (predominantly ABCG2 and ABCB1). The SP from CaLo cells showed an α6-integrin(bri)/CD(dim) pattern, whereas the SP from the SiHa cells showed an α6-integrin(-)/CD(dim) pattern. Recultured cells from the SPs of both cell lines generated both SPs and NSPs, and had higher clonogenic potential to form mainly holoclones and greater colony-forming efficiency under anchorage-independent growth conditions than the cells from the NSPs or total cell populations. Interestingly, we identified no SP in the HPV-uninfected C-33A cell line, and it did not express ABCG2 or other members of the ABC transporters (ABCB1, ABCC1, or ABCA3).

Twin births in Yucatan, Mexico during 2008-2020: trends in maternal sociodemographic factors and differences in birth weight and length according to sex of co-twin.

Background: Over the years, there has been a noticeable increase in the incidence of multiple pregnancies, due in part to the increased use of assisted reproductive technologies in recent years. The increase in twin pregnancies constitutes a public health challenge due to the adverse outcomes sometimes they entail. Twin pregnancies inherently carry a higher risk of complications, and one of the critical associated factors is the risk of low birth weight. Twin birth weight discordance among different populations can be attributed to differences in non-shared environmental influences. The presence of two or more fetuses in the uterus may lead to an unequal distribution of nutritional and oxygen resources, increasing the likelihood that at least one of the twins will experience insufficient fetal development. Other factors, such as ethnicity, genetics, sociodemographic characteristics, gestational age, parity, and chorion type, have also been related to the birth weight discordance in twin pregnancies. However, it is unclear to what extent the associations between these factors can explain the differences in birth and length size. The frequency of twin births varies among populations and over time, so it is crucial to gain a deeper understanding of the factors contributing to the rise in the twinning rate. The official records of twins continue to make significant contributions to our understanding of the causes of individual differences, and new twin registries are still being created. The recent availability of data from developing countries allows the analysis of trends in regions with sociodemographic and reproductive profiles. Obtaining a more comprehensive understanding of the epidemiology, as well as the related morbidity and mortality, is clinically crucial. Objectives: The aim of the study was to describe the trends of twin births in Yucatan, Mexico during 2008-2021, analyze their association with maternal sociodemographic factors, and compare birth outcomes between types of twin pairs: female-female, male-male, and female-male. Methods: A retrospective cohort analysis was conducted using data from the Mexican Ministry of Health, encompassing information on all births registered in the State of Yucatán, Mexico, from 2008 through 2021. The data was obtained from both public and private hospitals. The variables, including date of birth, sex, gestational age, birth weight and length of newborns, mother´s date of birth, educational level, and number of previous live offspring, were extracted from each dataset. Multiple births (three or more) were excluded from the study. We graphically analyzed the rates (per 1000 births) and percentages of twins according to maternal age, education level, and parity during the study period. A multiple logistic regression model was used to analyze the association between maternal sociodemographic factors and the occurrence of twin births. Comparisons of the gestational age and birth weight and length between types of twin pairs were performed using analysis of variance. Results: A total of 478,118 live births, including 1.4% twins (accounting 6,766 twin births), were analyzed. The rates increased from 11.21 during 2008-2011 to 13.34 during 2012-2017 and reached 20.08 in 2019. The percentages increased in women aged ≥30 years and those with higher educational levels. Older maternal age (coefficient = 0.03; OR = 1.03, per each year), greater education level (coefficient = 0.55 and OR= 1.74 for medium and coefficient = 1.05; OR = 2.57 for high level, compared with no education) and higher parity (coefficient= 0.26; OR = 1.30 per each previous offspring) increased the odds for having twins. Twins' male-male showed a slightly increased of preterm birth than a co-twin female. Opposite-sex twins showed measurable but small increases in birth weight and length compared with same-sex twins. Conclusion: The rate of twins in Yucatan increased substantially during 2008-2020 in specific sociodemographic groups. Opposite-sex twins were slightly larger than same-sex twins at birth.

Childhood acute lymphoblastic leukemia survival and spatial analysis of socio-environmental risks in Mexico.

Background: Acute lymphoblastic leukemia (ALL) etiology remains largely unknown; incidence patterns by age, sex, and geographical distribution suggest a potential environmental role. Aim: To identify ALL clusters from four contrasting urban areas of Mexico and to characterize the sources of environmental carcinogens. Methods: Hospital-based ALL cases (n = 443) diagnosed in children <19 years old from the Metropolitan Zones of Merida and San Luis Potosi, the State of Mexico, and Tijuana were analyzed (2015-2020). ALL cases were coded according to the International Classification of Diseases for Oncology. ALL clusters were identified by Kernel Density, and excess risk was estimated. Data of particulate matter ≤2.5 µm (PM2.5) concentrations measured by community-monitoring stations were analyzed. Geocoded datasets of benzene, polycyclic aromatic hydrocarbons, and PM2.5 sources were analyzed to characterize patterns of exposure in ALL clusters. Results: The survival rate for ALL ranged from 61.5% to 78.6%. Seven ALL clusters with excess risk (RR 1.4-2.3, p < 0.05) were identified. The carcinogen sources included artisanal brick kilns, gas stations, cement works, carpentry, paint, and chemical manufacturing establishments. PM2.5 levels ranged from 15 µg/m3 to 37 µg/m3 among study areas. Conclusion: ALL clusters were identified at the community level; the excess risk could be explained by small-scale carcinogen sources. The levels of PM2.5 in outdoor air ranged from 3 to 6 times above the World Health Organization (WHO) air quality guidelines. Healthcare providers must raise awareness of the increased risk of ALL in children living near sources of environmental carcinogens; cancer control and prevention strategies must be steered from a multi-sectoral and multi-action perspective to protect children's health.

Microwave-enhanced synthesis of 26-amino-22-oxocholestanes and their cytotoxic activity.

The synthesis of a series of 26-amino-22-oxocholestanes derived from diosgenin was accomplished via the substitution of an iodine atom at C-26 by primary and secondary amines. The reactions were conducted in refluxing acetonitrile and through microwave-assisted heating. The latter shows significant improvements in terms of reaction times going from hours to a few minutes or even seconds for completion. Only one of the selected amines, 4-aminourazole, did not yield the substitution product and the imine formation pathway was investigated instead, achieving the 26-iminourazole-22-oxocholestane. All the final products have been characterized and the cytotoxic activity of three of them has been evaluated in SiHa, MCF-7 and MDA tumor cell lines by the sulforhodamine B assay.

In vitro effects of stromal cells expressing different levels of Jagged-1 and Delta-1 on the growth of primitive and intermediate CD34(+) cell subsets from human cord blood.

In trying to contribute to our knowledge on the role of Notch and its ligands within the human hematopoietic system, we have assessed the effects of the OP9 stroma cell line - naturally expressing Jagged-1 - transduced with either the Delta-1 gene (OP9-DL1 cells) or with vector alone (OP9-V), on the in vitro growth of two different hematopoietic cell populations. Primitive (CD34(+) CD38(-) Lin(-)) and intermediate (CD34(+) CD38(+) Lin(-)) CD34(+) cell subsets from human cord blood were cultured in the presence of 7 stimulatory cytokines under four different conditions: cytokines alone (control); cytokines and mesenchymal stromal cells; cytokines and OP9-V cells; cytokines and OP9-DL1 cells. Proliferation and expansion were determined after 7days of culture. Culture of CD34(+) CD38(-) Lin(-) cells in the presence of OP9-V or OP9-DL1 cells resulted in a significant increase in the production of new CD34(+) CD38(-) Lin(-) cells (expansion), which expressed increased levels of Notch-1; in contrast, production of total nucleated cells (proliferation) was reduced, as compared to control conditions. In cultures of CD34(+) CD38(+) Lin(-) cells established in the presence of OP9-V or OP9-DL1 cells, expansion was similar to that observed in control conditions, whereas proliferation was also reduced. Interestingly, in these latter cultures we observed production of CD34(+) CD38(-) Lin(-) cells. Our results indicate that, as compared to MSC, OP9 cells were more efficient at inducing self-renewal and/or de novo generation of primitive (CD34(+) CD38(-) Lin(-)) cells, and suggest that such effects were due, at least in part, to the presence of Jagged-1 and DL1.

A Glycosaminoglycan-Rich Fraction from Sea Cucumber Isostichopus badionotus Has Potent Anti-Inflammatory Properties In Vitro and In Vivo.

Sea cucumber body wall contains several naturally occurring bioactive components that possess health-promoting properties. Isostichopus badionotus from Yucatan, Mexico is heavily fished, but little is known about its bioactive constituents. We previously established that I. badionotus meal had potent anti-inflammatory properties in vivo. We have now screened some of its constituents for anti-inflammatory activity in vitro. Glycosaminoglycan and soluble protein preparations reduced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammatory responses in HaCaT cells while an ethanol extract had a limited effect. The primary glycosaminoglycan (fucosylated chondroitin sulfate; FCS) was purified and tested for anti-inflammatory activity in vivo. FCS modulated the expression of critical genes, including NF-ĸB, TNFα, iNOS, and COX-2, and attenuated inflammation and tissue damage caused by TPA in a mouse ear inflammation model. It also mitigated colonic colitis caused in mice by dextran sodium sulfate. FCS from I. badionotus of the Yucatan Peninsula thus had strong anti-inflammatory properties in vivo.

Analysis of the dynamic aberrant landscape of DNA methylation and gene expression during arsenic-induced cell transformation.

Inorganic arsenic is a well-known carcinogen associated with several types of cancer, but the mechanisms involved in arsenic-induced carcinogenesis are not fully understood. Recent evidence points to epigenetic dysregulation as an important mechanism in this process; however, the effects of epigenetic alterations in gene expression have not been explored in depth. Using microarray data and applying a multivariate clustering analysis in a Gaussian mixture model, we describe the alterations in DNA methylation around the promoter region and the impact on gene expression in HaCaT cells during the transformation process caused by chronic exposure to arsenic. Using this clustering approach, the genes were grouped according to their methylation and expression status in the epigenetic landscape, and the changes that occurred during the cellular transformation were identified adequately. Thus, we present a valuable method for identifying epigenomic dysregulation.

Sustained improvement of motor function in hemiparkinsonian rats chronically treated with low doses of caffeine or trihexyphenidyl.

The effects of chronic oral treatment with low doses of caffeine (1-3 mg/kg) and trihexyphenidyl (0.1-0.2 mg/kg) were tested on hemiparkinsonian rats, which received the following treatments in a counterbalanced order: vehicle, caffeine, trihexyphenidyl, and caffeine plus trihexyphenidyl. Three preclinical models were used: the stepping test, the cylinder test, and the staircase test. Compared to pre-lesion values, the forepaw contralateral to the dopamine-denervated side showed impaired stepping, fewer wall contacts in the cylinder test, and fewer pellets retrieved in the staircase test. In the stepping test both doses of caffeine produced a complete recovery of motor function (100%), whereas the effect of trihexyphenidyl was less intense (77-80%). In this same test the maximal effect of drugs did not develop tolerance during 2-3 weeks, and was completely reversible after drug cessation. In the cylinder test only the wall contacts performed simultaneously with both forepaws were significantly increased by caffeine (3 mg/kg) and trihexyphenidyl (0.2 mg/kg), and this effect was also reversible. In the staircase test none of the treatments improved food pellet retrieval with the contralateral forepaw. Altogether, these results show that chronic treatment with caffeine, at doses similar to daily human consumption, produces a sustained improvement in the use of the contralateral forelimb in unilaterally 6-hydroxydopamine denervated rats, without the development of tolerance. Although the combined administration of caffeine plus trihexyphenidyl showed no synergism in these models, the results suggest that low doses of caffeine (1-3 mg/kg/day) could be of therapeutic value for the reversal of motor symptoms in parkinsonian patients.

A novel automated rat catalepsy bar test system based on a RISC microcontroller.

Catalepsy tests performed in rodents treated with drugs that interfere with dopaminergic transmission have been widely used for the screening of drugs with therapeutic potential in the treatment of Parkinson's disease. The basic method for measuring catalepsy intensity is the "standard" bar test. We present here an easy to use microcontroller-based automatic system for recording bar test experiments. The design is simple, compact, and has a low cost. Recording intervals and total experimental time can be programmed within a wide range of values. The resulting catalepsy times are stored, and up to five simultaneous experiments can be recorded. A standard personal computer interface is included. The automated system also permits the elimination of human error associated with factors such as fatigue, distraction, and data transcription, occurring during manual recording. Furthermore, a uniform criterion for timing the cataleptic condition can be achieved. Correlation values between the results obtained with the automated system and those reported by two independent observers ranged between 0.88 and 0.99 (P<0.0001; three treatments, nine animals, 144 catalepsy time measurements).

Selective A2A, but not A1 adenosine antagonists enhance the anticataleptic action of trihexyphenidyl in rats.

In rats made cataleptic with haloperidol (5.32 micromol/kg), the bar test was used to assess the possible synergism between the muscarinic antagonist trihexyphenidyl (THP) and selective adenosine A(1) and A(2A) receptor antagonists. Neither catalepsy intensity nor latency were affected by a subthreshold dose of THP (0.33 micromol/kg). The selective adenosine A(1) antagonist 8-cyclopentyl-1,3-dipropyl-xanthine (DPCPX) (5.15 micromol/kg) caused a small, but significant reduction of catalepsy intensity that remained unchanged when combined with THP. DPCPX had no effect on catalepsy latency, either alone or combined with THP. In contrast, an equimolar dose of the selective adenosine A(2A) antagonist 4-(2-[7-amino-2-(2-furyl)1,2,4-triazolo[2,3-a]-[1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) (5.15 micromol/kg) produced a significant reduction of catalepsy intensity and increased catalepsy latency. Both effects were potentiated when ZM 241385 was combined with THP. The synergism was more evident when rats were pretreated with a subthreshold dose of ZM 241385 (1.55 micromol/kg) that was unable to modify catalepsy parameters when applied alone, but produced a significant reduction in catalepsy intensity and an increase in catalepsy latency when administered with THP. Catalepsy was unaffected by a combination of equimolar, subthreshold doses of DPCPX (1.55 micromol/kg) and ZM 241385 (1.55 micromol/kg). These findings indicate that the anticataleptic effect of anticholinergics is enhanced only by the selective blockade of adenosine A(2A) receptors.

Treatment with subthreshold doses of caffeine plus trihexyphenidyl fully restores locomotion and exploratory activity in reserpinized rats.

Trihexyphenidyl (THP) is a drug commonly used to reduce parkinsonian symptoms. An important side effect of this agent is memory impairment. Since caffeine enhances the potency of THP to inhibit haloperidol-induced catalepsy, caffeine may be used as an adjuvant of lower doses of THP, in order to improve its antiparkinsonian effects without causing memory disruption. To further assess the synergism between caffeine and THP, both drugs were tested in reserpinized rats, another preclinical model of Parkinson's disease. Four groups of rats (n = 7) were treated with reserpine (5 mg/kg, i.p.). A control group (n = 7) was treated only with the vehicle for reserpine (dimethylsulphoxide). The spontaneous locomotor behavior was tested 24 h later in a box with infrared sensors, 30 min after receiving one of the following treatments: distilled water (1 ml/kg), caffeine (1 mg/kg), THP (0.1 mg/kg) or caffeine plus THP. The levels of horizontal locomotion (14 +/- 5%) and vertical exploration (15 +/- 10%) were significantly lower in reserpinized rats treated with distilled water, compared with the mean activity values (100%) recorded in animals pretreated only with the vehicle for reserpine. The reserpine-induced hypokinesia was neither reversed by caffeine alone nor by THP alone. However, the combination of caffeine plus THP restored locomotion (141 +/- 19%) and vertical exploration (82 +/- 17%) to levels not significantly different to those of non-reserpinized rats. Moreover, the time-course of locomotion and exploration displayed the characteristic habituation over time, in which short-term memory processes are involved. Also, the thigmotaxis index indicated that the combined treatment did not induce anxiety-like behavior. Hence, these results support the proposal that low, subthreshold doses of caffeine plus THP have the potential to alleviate the motor disabilities in parkinsonian patients, with a low risk of causing anxiety or memory impairment.

Long-lasting resistance to haloperidol-induced catalepsy in male rats chronically treated with caffeine.

Chronic caffeine consumption has been inversely associated with the risk of developing Parkinson's disease. Here we assessed whether chronic caffeine treatment increases the resistance of male Wistar rats to haloperidol (1mg/kg, s.c.)-induced catalepsy, measured in the bar test at 15 min intervals during 3h. Caffeine (5mg/kg/day) was delivered for 6 months via drinking water. Control rats received only tap water. Treatments began when animals were 3-4 months old. In order to unveil long-lasting catalepsy refractoriness not attributable to the presence of caffeine in the brains of rats, they were evaluated from day 18 to day 27 after caffeine withdrawal, a time that is far in excess for the full excretion of a caffeine dose in this species. The average cataleptic immobility measured in caffeine-treated rats (n=23) was 1148+/-140 s, a value 34+/-8% lower than that recorded in control animals (n=20), whose mean immobility was 1736+/-137 s (P=0.0026, t-test). The percentage of catalepsy reduction measured in caffeine-treated rats evaluated on days 18-20 after caffeine discontinuation (-32+/-13%, n=12, P<0.05) was comparable to the catalepsy decrease recorded in those animals tested on days 21-27 (-36+/-10%, n=11, P<0.02), a finding compatible with the notion that the effect was indeed mediated by enduring changes of brain functioning and not by the physical presence of caffeine or its metabolites. Caffeine-treated rats also had higher catalepsy latency scores compared with control rats (P<0.01, U-test). The present findings show that chronic consumption of caffeine produces perdurable resistance to catalepsy induced by dopamine receptor blockade, possibly through enhancement of dopamine transmission, giving further support to the epidemiological results indicating that prolonged caffeine consumption affords neuroprotection against Parkinson's disease.