Loneliness and perceived social support in pregnancy and early postpartum of mothers living with HIV in Ontario, Canada.

The HIV Mothering Study (n = 72) was a prospective, observational, cohort study exploring psychosocial experiences and needs of WLWHIV in pregnancy and postpartum. We performed quantitative analysis of determinants of loneliness (UCLA Loneliness Scale) and lower perceived social support (SS) (Medical Outcomes Study-Social Support Survey). The hypothesized determinants included: age, years with HIV, racism (Everyday Discrimination Scale), depression (Edinburgh Postnatal Depression Scale [EPDS]), nadir CD4 (<200 cells/µL), tertiary vs. community HIV care, and marital status. The median age was 33 (IQR = 30-37); 65.3% were African/Caribbean/Black. Multivariable analyses revealed associations between marital status and perceived social support (ß = -16.48, p < 0.0001), and this association was also seen with change over time (p = 0.02). Variables associated with SS that did not change over time were: income, EDS racism, EPDS score. Significant associations with loneliness were seen with the same variables associated with SS. Variables associated with loneliness that also changed over time were: EDS Racism (ß = 0.22, p = 0.0005, and over time p = 0.003), and EPDS score (ß = 0.74, p < 0.0001), and over time (p = 0.0211). Variables associated with loneliness but that did not change over time were: marital status and income. This analysis provides clinicians with prenatal risk factors which may be associated with increase loneliness and lower SS during pregnancy and postpartum: marital status, income, racism and depression.

Strategies to modulate BHK cell proliferation by the regulation of IRF-1 expression.

Activation of the constitutively expressed interferon-regulatory-factor-1/estrogen receptor fusion protein (IRF-1-hER) in BHK cells was accomplished through the addition of estradiol to the culture medium, which enabled IRF-1 to gain its transcriptional activator function and inhibit cell growth. With the addition of 100 nM estradiol at the beginning of the exponential phase of a cell suspension culture, IRF-1 activation led to a rapid cell growth inhibition but also to a significant decrease in cell viability. To apply this concept in industry, a reduction of the time span of estradiol exposure is required. Cycles of estradiol addition and removal were performed in 2-l stirred tank bioreactors operated under perfusion, where an initial step addition of 100 nM estradiol was performed, followed, after 48-72 h, by a slow dilution with estradiol-free fresh medium (perfusion rate varying between 0.7 and 1.4 per day). Cell growth inhibition was successfully achieved for three consecutive cycles. Diluting the estradiol by perfusing medium without estradiol to concentrations lower than 10 nM led to cell growth and viability recovery independently of the perfusion rate used. These observations permitted the definition of operational strategies for regulated IRF-1 BHK cell growth by pulse estradiol addition, followed by a period of 48 h in the presence of estradiol and by fast perfusion to estradiol concentrations lower than 10 nM. Cell growth response to IRF-1 activation and following estradiol removal by perfusion was also evaluated with an IRF-1-hER regulated clone expressing constitutively Factor VII, where the time of estradiol exposure and perfusion rate were varied. This clone presented a stronger response to IRF-1 activation without an increase in Factor VII specific productivity after cell growth inhibition; this clearly indicates that the stationary phase obtained is clone dependent. This work proves that it is possible to modulate the IRF-1 effect for cell growth control by the manipulation of cycles of addition and removal of estradiol, potentially representing a new generation of culture procedures for controlled growth production purposes.

Metabolic changes during cell growth inhibition by p27 overexpression.

The overexpression of p27, a cyclin-dependent kinase (CDK) inhibitor, has been shown to effectively inhibit cell growth at the G1-phase of different cell lines, potentiating a valid genetic strategy for cell proliferation control. In order to characterize the energy requirements after p27 overexpression in CHO cells expressing SEAP (secreted form of the human alkaline phosphatase enzyme), key metabolic parameters were evaluated. Cell growth inhibition led to a significant increase in cell size concomitant with a 2-fold increase in cell protein content. The simultaneous increase of the intracellular proteolytic activity with protein content suggests higher protein synthesis. A general 2-fold increase in oxygen, glutamine and glucose consumption rates, coupled with an increase in lactate and ammonia production was observed. p27 overexpression led to a significant increase in the intracellular pool of AMP (8.5-fold), ADP (6-fold) and, more uncommonly, ATP (4.5-fold). Nevertheless, cells were able to maintain the equilibrium among the three adenine nucleotides since both the ATP/ADP ratio and the energy charge values remained similar to those observed with non-growth inhibited cells. This work shows that the observed 4-fold increase in SEAP specific productivity after cell growth inhibition by p27, occurred concomitantly with a higher expenditure of cell energy. This characterization of cell metabolism becomes important in demonstrating the applicability of growth inhibition systems.

Manipulation of culture conditions for BHK cell growth inhibition by IRF-1 activation.

The activation of interferon-regulatory-factor-1 (IRF-1) hasbeen applied to regulate the cell growth of BHK cells. Theconstitutively expressed IRF-1-estrogen receptor fusion protein(IRF-1-hER) activated by the addition to the culture medium ofan estrogen analogue (estradiol), enabled IRF-1 to gain itstranscriptional activator function. By using a dicistronicstabilised self-selecting construct it was possible to controlcell proliferation. With the addition of 100 nM of estradiol at the beginning of the exponential phase, the IRF-1 activationled to a rapid cell growth inhibition. Two days after estradioladdition cell concentration was still maintained but a decreasein cell viability was observed. This cell response isindependent on clone (producer and non-producer) and culturesystem (static and stirred cultures). Specificrecombinant-protein productivity of the producer clone was notsignificantly altered. Control experiments confirmed that IRF-1activation effect was not due to the addition of estradiol per se, estradiol solvent or serum concentration. The extent ofcell growth inhibition is dependent on estradiol concentrationand estradiol addition time, although a decrease in cellviability was always observed. Reducing the time span ofestradiol exposure allowed the decrease in the cell viability tobe controlled and the stationary inhibited phase to be extended:when the time of contact between the cells and estradiol isreduced cell viability increases, archieving values similar tothose obtained if no estradiol is added. During this recoveryphase the cells passed two different phases: first a stationaryphase extension where cell growth was still inhibited, followedby an increase of cell concentration. The IRF-1 system isreversible. This pattern can be repeated for an extended period when estradiol addition and removal are repeated, showing acyclic response. Thus, it is possible to modulate the IRF-1effect by manipulating cycles of addition/removal of estradioland in this way the stationary phase can be maintained.