Human liver cell spheroids in extended perfusion bioreactor culture for repeated-dose drug testing

Primary cultures of human hepatocyte spheroids are a promising in vitro model for long-term studies of hepatic metabolism and cytotoxicity. The lack of robust methodologies to culture cell spheroids, as well as a poor characterization of human hepatocyte spheroid architecture and liver-specific functionality, have hampered a widespread adoption of this three-dimensional culture format. In this work, an automated perfusion bioreactor was used to obtain and maintain human hepatocyte spheroids. These spheroids were cultured for 3-4 weeks in serum-free conditions, sustaining their phase I enzyme expression and permitting repeated induction during long culture times; rate of albumin and urea synthesis, as well as phase I and II drug-metabolizing enzyme gene expression and activity of spheroid hepatocyte cultures, presented reproducible profiles, despite basal interdonor variability (n = 3 donors). Immunofluorescence microscopy of human hepatocyte spheroids after 3-4 weeks of long-term culture confirmed the presence of the liver-specific markers, hepatocyte nuclear factor 4α, albumin, cytokeratin 18, and cytochrome P450 3A. Moreover, immunostaining of the atypical protein kinase C apical marker, as well as the excretion of a fluorescent dye, evidenced that these spheroids spontaneously assemble a functional bile canaliculi network, extending from the surface to the interior of the spheroids, after 3-4 weeks of culture. Conclusion: Perfusion bioreactor cultures of primary human hepatocyte spheroids maintain a liver-specific activity and architecture and are thus suitable for drug testing in a long-term, repeated-dose format.     

In vitro restoration of Th17 response during HIV infection with an antiretroviral drug and Th17 differentiation cytokines

The Th17 subset is preferentially depleted as compared to the Th1 subset in chronically HIV-infected patients, even after successful antiretroviral therapy. In this study, we have established an in vitro system utilizing primary human CD4 T cell cultures that recapitulates the dramatic loss of Th17 response upon HIV-1 infection that is accompanied with a less profound Th1 decrease. With this experimental system, we showed that blocking viral entry with CCR5 ligands or TAK779 reduced the infection and enhanced Th17 response but not Th1 response. Antiretroviral drug 3TC (lamivudine), given at the time of infection, completely prevented the loss of Th17 and Th1 responses but was ineffective when given after infection was already established. Only when Th17 differentiation cytokines were given along with 3TC to the cultures with established HIV infection was Th17 response fully restored and virus replication kept suppressed. Finally, a significant increase of Th17 response was achieved in peripheral lymphocytes of HIV-infected patients on antiretroviral therapy after treatment with Th17 differentiation cytokines. These data demonstrate the presence of CD4 T cells remaining capable of mounting Th17 response during HIV infection and indicate the potential use of immunotherapeutic modalities to supplement antiretroviral drugs for restoring Th17 response in chronically HIV-infected patients.     

Predicting retinal pigment epithelium remodelling and its functional impact

Graefe's Archive for Clinical and Experimental Ophthalmology - To identify predictive factors for RPE tear remodelling and its correlation with functional and morphological outcomes....     

Pudendal nerve block in HDR-brachytherapy patients: do we really need general or regional anesthesia?

Purpose

In male patients, the pudendal block was applied only in rare cases as a therapy of neuralgia of the pudendal nerve. We compared pudendal nerve block (NPB) and combined spinal-epidural anesthesia (CSE) in order to perform a pain-free high-dose-rate (HDR) brachytherapy in a former pilot study in 2010. Regarding this background, in the present study, we only performed the bilateral perineal infiltration of the pudendal nerve.

Methods

In 25 patients (71.8 ± 4.18 years) suffering from a high-risk prostate carcinoma, we performed the HDR-brachytherapy with the NPB. The perioperative compatibility, the subjective feeling (German school marks principle 1–6), subjective pain (VAS 1–10) and the early postoperative course (mobility, complications) were examined.

Results

All patients preferred the NPB. There was no change of anesthesia form necessary. The expense time of NPB was 10.68 ± 2.34 min. The hollow needles (mean 24, range 13–27) for the HDR-brachytherapy remained on average 79.92 ± 12.41 min. During and postoperative, pain feeling was between 1.4 ± 1.08 and 1.08 ± 1.00. A transurethral 22 French Foley catheter was left in place for 6 h. All patients felt the bladder catheter as annoying, but they considered postoperative mobility as more important as complete lack of pain. The subjective feeling was described as 2.28 ± 0.74. Any side effects or complications did not appear.

Conclusions

Bilateral NPB is a safe and effective analgesic option in HDR-brachytherapy and can replace CSE. It offers the advantage of almost no impaired mobility of the patient and can be performed by the urologist himself. Using transrectal ultrasound guidance, the method can be learned quickly.     

Chloroquine-induced hyperpigmentation of the hard palate

This article reports a rare case of extensive palatal pigmentation secondary to long-term chloroquine treatment. Chloroquine was originally used as an antimalarial agent, but it is now widely used as an adjunct in the treatment of autoimmune diseases. Adverse effects of chloroquine usually include skin changes such as bullous pemphigoid, exacerbation of psoriasis, and pigmentation of the skin and mucous membranes as well as retinopathy, gastrointestinal alterations, and neuromuscular disorders. Extensive oral pigmentation is an uncommon feature of an adverse drug effect, and diagnosis should be based on clinicopathological findings.     

Application of forensic anthropology to non-forensic issues: an experimental taphonomic approach to the study of human body decomposition in aerobic conditions

The knowledge of the decomposition process takes a progressively central role in cemetery management. In Europe there is an increase in constructing concrete modules that allow aerobic decomposition, which, theoretically, have advantages. By allowing the circulation of oxygen they will lead to a rapid decomposition, and, consequently, permit burial space rotation. This method also allows the burial of several corpses in small areas. In addition, it solves environmental issues. However, the ways in which the decomposition occurs under these conditions and the time required for skeletonization are still unknown. The aim of this study is to analyse the changes of the cadavers buried in aerobic module systems, realize the time necessary for skeletonization, and study solutions that might enable a faster decomposition. This experimental study utilized three aerobic module systems already used in Portuguese cemeteries and a prototype, with four pig carcasses as animal models. The evolution of the decomposition process was assessed weekly, over 147 days (2184 ADD). This study showed differences in the decomposition process between the four systems. The results allowed us to select the necessary characteristics for the construction of aerobic modules enabling faster decomposition.     

Fathers’ parenting self-efficacy during the transition to parenthood

Background: Little is known about the development of fathers’ parenting self-efficacy during the transition to parenthood.

Objectives: To analyse (1) fathers’ parenting self-efficacy developmental path and (2) the effects of anxious and depressive symptoms and coparenting support on fathers’ parenting self-efficacy developmental path, from the first trimester of pregnancy to 6 months postpartum.

Methods: Eighty-six fathers recruited at the first trimester of pregnancy completed self-report measures of anxious and depressive symptoms, coparenting support and parenting self-efficacy at the first and third trimesters of pregnancy, and at 1 and 6 months postpartum. Growth curve models were performed.

Results: An increase in fathers’ parenting self-efficacy was found from the first trimester of pregnancy to 6 months postpartum. The main effects of anxious symptoms and interaction effects of anxious symptoms and time were found on fathers’ parenting self-efficacy. Fathers with higher anxious symptoms revealed lower levels of parenting self-efficacy at the first trimester of pregnancy and a lower increase of parenting self-efficacy from this time to 6 months postpartum. The main effects of coparenting support were found in fathers’ parenting self-efficacy. At the first trimester of pregnancy, fathers who perceived more coparenting support revealed higher levels of parenting self-efficacy.

Conclusion: The present study may contribute to the literature by describing fathers’ parenting self-efficacy developmental path and the effects of anxious symptoms and coparenting support on fathers’ parenting self-efficacy developmental path during the transition to parenthood.     

Multiplex PCR strategy for subtyping the staphylococcal cassette chromosome mec type IV in methicillin-resistant Staphylococcus aureus: 'SCCmec IV multiplex'

OBJECTIVES: To develop and validate a new multiplex PCR strategy for subtyping SCCmec type IV methicillin-resistant Staphylococcus aureus (MRSA) strains-SCCmec IV multiplex PCR. METHODS: Seven primer pairs were designed to detect the ccrB allotype 2 (internal positive control), the five polymorphic J1 regions described so far for SCCmec type IV and the new J1 region specific for EMRSA-15. Primer sets were tested for specificity and robustness with prototype strains for each subtype of SCCmec type IV. The multiplex PCR conditions were optimized in a trial-error approach. RESULTS: The seven prototype strains for the earlier described subtypes of SCCmec type IV and the EMRSA-15 prototype strain were correctly characterized by our strategy. Moreover, 13 diverse SCCmec type IV strains could be assigned to a subtype of SCCmec type IV and 5 EMRSA-15 strains were assigned to the new subtype IVh. One strain could not be assigned to an SCCmec type IV subtype because of the absence of amplification of the specific J1 region. CONCLUSIONS: This new strategy, based on a single multiplex PCR reaction, is adequate for the rapid assignment of all major subtypes of SCCmec type IV described so far and also the new subtype IVh characteristic of EMRSA-15. This strategy complements well the previously described multiplex PCR assay for the rapid assignment of SCCmec types.