DR antigen expression on ovarian carcinoma cells does not correlate with their capacity to elicit an autologous proliferative response

Summary Expression of HLA-DR antigens by purified preparations of human ovarian carcinoma cells freshly isolated from surgical specimens was examined in parallel with the capacity of tumor cells to elicit a blastogenic response from autologous lymphocytes in mixed lymphocyte-tumor culture (MLTC) assay. Of 21 tumor preparations, 11 (52%) reacted with monoclonal antibodies 279 and/or 949 specific for a monomorphic determinant of HLA-DR antigens, with heterogeneous positivity, ranging between 30% and 95%. In this series of patients positive MLTC occurred in 8/21 individual experiments. The HLA-DR expression was proportionally similar in tumors giving positive MLTC (4/8=50%) and negative MLTC (7/13=53%). The lack of correlation between DR expression on tumor cells and stimulatory activity in autologous MLTC and the fact that DR-negative tumors could induce lymphocyte stimulation, support the hypothesis that blastogenesis occurs upon recognition of tumor-associated antigens, different from DR molecules, possibly tumor-specific antigens.     

Decline in measles mortality: nutrition,age at infection,or exposure?

The mortality from measles was studied in an urban area of Guinea-Bissau one year before and five years after the introduction of a vaccination programme. The years after the introduction of immunisation saw a decline in mortality among unvaccinated children with measles. This decline occurred despite a lower age at infection and an increasing prevalence of malnourished children. State of nutrition (weight for age) did not affect the outcome of measles infection. The incidence of isolated cases, however, increased in the period after the introduction of measles vaccination. As mortality was lower among these cases, diminished clustering explained some of the reduction in mortality. Comparison between the urban district and a rural area inhabited by the same ethnic group showed a lower age at infection, less clustering of cases, and lower case fatality ratios in the urban area.Endemic transmission of measles in urban districts leads to less clustering of cases, which may help explain the usually lower case fatality ratios in these areas. As measles vaccination increases herd immunity and diminishes clustering of cases, it may reduce mortality even among unvaccinated children who contract the disease.     

In search of specific cytotoxic T lymphocytes infiltrating or accompanying human ovarian carcinoma

Summary Lymphocytes infiltrating human ovarian carcinoma obtained directly from the tumour mass (tumour-infiltrating lymphocytes, TIL) or from the carcinomatous ascites (tumour-associated lymphocytes, TAL) were expanded in vitro in long-term cultures with interleukin-2 and tested for their specific cytolytic activity. Killing of the autologous tumour was detected only in a proportion of the patients, less frequently in TIL compared to TAL. In fact two out of ten TIL and four out of nine TAL cultures tested showed significant levels of lysis against the autologous tumour. This cytotoxic activity was not restricted to the autologous tumour, as other tumour cell lines, including non-ovarian ones, were lysed as well. The cultures that were not cytotoxic against the autologous tumour were in most cases able to lyse other tumour cell lines of ovarian or other histology. Cloning of TIL from one patient was performed: of 22 clones tested, 4 displayed higher cytotoxicity against the autologous tumour compared to the uncloned population and 3 out of these 4 did not kill an irrelevant carcinoma cell line. In order to stimulate the expansion of putative specific effectors we performed mixed lymphocyte/tumour cultures (MLTC) with autologous or allogeneic tumour cells. No stimulation of cytotoxicity against the autologous tumour was detected after MLTC in nine different TAL populations, using autologous or allogeneic tumours as stimulators. On the contrary, peripheral blood lymphocytes from two patients after MLTC with the autologous tumour showed increased killing of the autologous and decreased killing of an allogeneic target. In conclusion TIL and TAL from ovarian carcinoma expanded in vitro with interleukin-2 usually have non-MHC-restricted cytotoxicity and variable degrees of reactivity against the autologous tumour. A preferential killing for the autologous tumour was not observed even after MLTC. These results do not exclude the existence of tumour-specific cytotoxic T lymphocytes in ovarian carcinoma; nevertheless they suggest that putative specific effectors have very low frequency and that culture techniques for expanding their growth more selectively are still to be optimized.     

Genotoxicity testing of chloramphenicol in rodent and human cells

The results of this work, carried out to extend the limited information at present available on the genotoxic potential of chloramphenicol (CAP), indicate that in millimolar concentrations this antibacterial agent produced a minimal amount of DNA fragmentation in both V79 cells and metabolically competent rat hepatocytes. Moreover, a level of DNA-repair synthesis indicative of a weak but positive response was detected in primary cultures of liver cells obtained from 2 of 3 human donors, and a borderline degree of repair was present in those prepared from rats. The promutagenic character of CAP-induced DNA lesions was confirmed by a low but significant increase in the frequency of 6-thioguanine-resistant clones of V79 cells, which, however, was absent when the exposure was done in the presence of co-cultured rat hepatocytes. Finally, oral administration to rats of 1/2 LD50 CAP did not increase the incidence of either micronucleated polychromatic erythrocytes or micronucleated hepatocytes. Taken as a whole these findings suggest that CAP should be considered a compound intrinsically capable of producing a very weak genotoxic effect, but only at concentrations about 25 times higher than those occurring in patients treated with maximal therapeutic dosages.     

Diversity of dermal fibroblasts as major determinant of variability in cell reprogramming

Induced pluripotent stem cells (iPSCs) are adult somatic cells genetically reprogrammed to an embryonic stem cell‐like state. Notwithstanding their autologous origin and their potential to differentiate towards cells of all three germ layers, iPSC reprogramming is still affected by low efficiency. As dermal fibroblast is the most used human cell for reprogramming, we hypothesize that the variability in reprogramming is, at least partially, because of the skin fibroblasts used. Human dermal fibroblasts harvested from five different anatomical sites (neck, breast, arm, abdomen and thigh) were cultured and their morphology, proliferation, apoptotic rate, ability to migrate, expression of mesenchymal or epithelial markers, differentiation potential and production of growth factors were evaluated in vitro. Additionally, gene expression analysis was performed by real‐time PCR including genes typically expressed by mesenchymal cells. Finally, fibroblasts isolated from different anatomic sites were reprogrammed to iPSCs by integration‐free method. Intriguingly, while the morphology of fibroblasts derived from different anatomic sites differed only slightly, other features, known to affect cell reprogramming, varied greatly and in accordance with anatomic site of origin. Accordingly, difference also emerged in fibroblasts readiness to respond to reprogramming and ability to form colonies. Therefore, as fibroblasts derived from different anatomic sites preserve positional memory, it is of great importance to accurately evaluate and select dermal fibroblast population prior to induce reprogramming.     

The JAK1/2 inhibitor ruxolitinib delays premature aging phenotypes

Hutchinson–Gilford progeria syndrome (HGPS) is caused by an LMNA mutation that results in the production of the abnormal progerin protein. Children with HGPS display phenotypes of premature aging and have an average lifespan of 13 years. We found earlier that the targeting of the transmembrane protein PLA2R1 overcomes senescence and improves phenotypes in a mouse model of progeria. PLA2R1 is regulating the JAK/STAT signaling, but we do not yet know whether targeting this pathway directly would influence cellular and in vivo progeria phenotypes. Here, we show that JAK1/2 inhibition with ruxolitinib rescues progerin‐induced cell cycle arrest, cellular senescence, and misshapen nuclei in human normal fibroblasts expressing progerin. Moreover, ruxolitinib administration reduces several premature aging phenotypes: bone fractures, bone mineral content, grip strength, and a trend to increase survival in a mouse model of progeria. Thus, we propose that ruxolitinib, an FDA‐approved drug, should be further evaluated as a drug candidate in HGPS therapy.     

Unbiased proteomic profiling of host cell extracellular vesicle composition and dynamics upon HIV‐1 infection

Cells release diverse types of extracellular vesicles (EVs), which transfer complex signals to surrounding cells. Specific markers to distinguish different EVs (e.g. exosomes, ectosomes, enveloped viruses like HIV) are still lacking. We have developed a proteomic profiling approach for characterizing EV subtype composition and applied it to human Jurkat T cells. We generated an interactive database to define groups of proteins with similar profiles, suggesting release in similar EVs. Biochemical validation confirmed the presence of preferred partners of commonly used exosome markers in EVs: CD81/ADAM10/ITGB1, and CD63/syntenin. We then compared EVs from control and HIV‐1‐infected cells. HIV infection altered EV profiles of several cellular proteins, including MOV10 and SPN, which became incorporated into HIV virions, and SERINC3, which was re‐routed to non‐viral EVs in a Nef‐dependent manner. Furthermore, we found that SERINC3 controls the surface composition of EVs. Our workflow provides an unbiased approach for identifying candidate markers and potential regulators of EV subtypes. It can be widely applied to in vitro experimental systems for investigating physiological or pathological modifications of EV release.     

FSHIP2 regulates epithelial cell polarity through its lipid product,which binds to Dlg1, a pathway subverted by hepatitis C virus core protein

The main targets of hepatitis C virus (HCV) are hepatocytes, the highly polarized cells of the liver, and all the steps of its life cycle are tightly dependent on host lipid metabolism. The interplay between polarity and lipid metabolism in HCV infection has been poorly investigated. Signaling lipids, such as phosphoinositides (PIs), play a vital role in polarity, which depends on the distribution and expression of PI kinases and PI phosphatases. In this study, we report that HCV core protein, expressed in Huh7 and Madin–Darby canine kidney (MDCK) cells, disrupts apicobasal polarity. This is associated with decreased expression of the polarity protein Dlg1 and the PI phosphatase SHIP2, which converts phosphatidylinositol 3,4,5-trisphosphate into phosphatidylinositol 4,5-bisphosphate (PtdIns(3,4)P2). SHIP2 is mainly localized at the basolateral membrane of polarized MDCK cells. In addition, PtdIns(3,4)P2 is able to bind to Dlg1. SHIP2 small interfering RNA or its catalytically dead mutant disrupts apicobasal polarity, similar to HCV core. In core-expressing cells, RhoA activity is inhibited, whereas Rac1 is activated. Of interest, SHIP2 expression rescues polarity, RhoA activation, and restricted core level in MDCK cells. We conclude that SHIP2 is an important regulator of polarity, which is subverted by HCV in epithelial cells. It is suggested that SHIP2 could be a promising target for anti-HCV treatment.