Culture-expanded human dermal stem cells exhibit donor to donor differences in cAMP generation

Stem cell techniques have facilitated a number of potential uses for such cells in cell therapy and drug development. Studies of the cAMP/protein kinase A (PKA) pathway are widely employed to investigate the effects of a large variety of substances. We assayed the cAMP pathway in human skin-derived mesenchymal stem cells (S-MSC) to evaluate donor to donor variations in response to pharmacological manipulations in vitro. Immunophenotyping of S-MSC revealed that, in general, 95% of S-MSCs were positive for CD90, CD73 and CD105 and negative for the expression of haemopoetic markers CD14, CD45 and human leukocyte antigen-DR (HLA-DR). Nevertheless, fluctuations occurred in basal cAMP levels from 5 pmol/mg to 18 pmol/mg. Total cAMP response element binding protein (CREB) concentrations ranged from 0.8 ng/ml to 1 ng/ml, whereas the proportions of phospho-CREB versus total CREB differed between the cell lines. Basic fibroblast growth factor (FGF-2) and epidermal growth factor (EGF) stimulated cAMP generation, whereas leukaemia inhibiting factor reduced some of their effects. Forskolin (0.05 and 1 mM) acted in synergy with FGF-2 and EGF; however, it caused pronounced donor to donor differences in the increase of cAMP and phospho-CREB levels. Additionally, dibutyryl-cAMP caused significant donor to donor variations in cell proliferation, possibly indicating a change of cell differentiation status. We speculate that similar donor diversity might be observed after cell stimulation with various G_s-protein-coupled receptor ligands. Heterogeneity of donor cell responses to stimulation of the cAMP pathway indicates the need for wide safety margins for S-MSC use in drug screening; nevertheless, knowledge of this heterogeneity might be useful for the design of donor-specific cell therapy.     

HIV-1 Specific Antibody Titers and Neutralization among Chronically Infected Patients on Long-Term Suppressive Antiretroviral Therapy (ART): A Cross-Sectional Study

The majority of potent and broadly neutralizing antibodies against HIV-1 have been isolated from untreated patients with acute or chronic infection. To assess the extent of HIV-1 specific antibody response and neutralization after many years of virologic suppression from potent combination ART, we examined antibody binding titers and neutralization of 51 patients with chronic HIV-1 infection on suppressive ART for at least three years. In this cross-sectional analysis, we found high antibody titers against gp120, gp41, and the membrane proximal external region (MPER) in 59%, 43%, and 27% of patients, respectively. We observed significantly higher endpoint binding titers for gp120 and gp41 for patients with >10 compared to ≤10 years of detectable HIV RNA. Additionally, we observed higher median gp120 and gp41 antibody titers in patients with HIV RNA <50 copies/mL for ≤5 years. 22% of patients neutralized a HIV-1 primary isolate (HIV-1_JR-FL) and 8% neutralized a HIV-2/HIV-1 MPER chimera. Significantly greater HIV-1_JR-FL neutralization was found among patients with >10 years of detectable HIV RNA (8/20 [40.0%] versus 3/31 [9.7%] for ≤10 years, p = 0.02) and a trend toward greater neutralization in patients with ≤5 years of HIV RNA <50 copies/mL (7/20 [35.0%] versus 4/31 [12.9%] for >5 years, p = 0.08). All patients with neutralizing activity mediated successful phagocytosis of VLPs by THP-1 cells after antibody opsonization. Our findings of highly specific antibodies to several structural epitopes of HIV-1 with antibody effector functions and neutralizing activity after long-term suppressive ART, suggest continuous antigenic stimulation and evolution of HIV-specific antibody response occurs before and after suppression with ART. These patients, particularly those with slower HIV progression and more time with detectable viremia prior to initiation of suppressive ART, are a promising population to identify and further study functional antibodies against HIV-1.     

Mitochondrial DNA origins of the Latvian clefting population

Latvia has one of the highest prevalence of isolated cleft lip with or without cleft palate (CL/P) in Europe. To clarify the genetic origins of the Latvian cleft population and establish a method for genetic mapping, mitochondrial DNA variation was studied in a population affected with clefting. One-hundred and seven subjects and 351 samples from unrelated healthy volunteers representing four anthropologically, archaeologically and ethno-linguistically different regions of Latvia were selected. The case group showed a higher frequency of haplogroups U4 (p=0.02) and U5 (p=0.0003) than in non-U haplogroups. We hypothesize that U4 and U5 mtDNA haplotype carriers may also carry susceptibility genes for clefts. Future studies will take into consideration these definitions based on mtDNA haplotypes when analyzing genetic variations and their possible contribution to CL/P susceptibility.     

Serum opacity factor unmasks human plasma high-density lipoprotein instability via selective delipidation and apolipoprotein A-I desorption

Human plasma high-density lipoproteins (HDL) are important vehicles in reverse cholesterol transport, the cardioprotective mechanism by which peripheral tissue-cholesterol is transported to the liver for disposal. HDL is the target of serum opacity factor (SOF), a substance produced by Streptococcus pyogenes that turns mammalian serum cloudy. Using a recombinant (r) SOF, we studied opacification and its mechanism. rSOF catalyzes the partial disproportionation of HDL into a cholesteryl ester-rich microemulsion (CERM) and a new HDL-like particle, neo HDL, with the concomitant release of lipid-free (LF)-apo A-I. Opacification is unique; rSOF transfers apo E and nearly all neutral lipids of approximately 100,000 HDL particles into a single large CERM whose size increases with HDL-CE content (r approximately 100-250 nm) leaving a neo HDL that is enriched in PL (41%) and protein (48%), especially apo A-II. rSOF is potent; within 30 min at 37 degrees C, 10 nM rSOF opacifies 4 microM HDL. At respective low and high physiological HDL concentrations, LF-apo A-I is monomeric and tetrameric. CERM formation and apo A-I release have similar kinetics suggesting parallel or rapid sequential steps. According to the reaction products and kinetics, rSOF is a heterodivalent fusogenic protein that uses a docking site to displace apo A-I and bind to exposed CE surfaces on HDL; the resulting rSOF-HDL complex recruits additional HDL with its binding-delipidation site and through multiple fusion steps forms a CERM. rSOF may be a clinically useful and novel modality for improving reverse cholesterol transport. With apo E and a high CE content, CERM could transfer large amounts of cholesterol to the liver for disposal via the LDL receptor; neo HDL is likely a better acceptor of cellular cholesterol than HDL; LF-apo A-I could enhance efflux via the ATP-binding casette transporter ABCA1.     

Characterization of a <Emphasis Type="Italic">Pinus sylvestris</Emphasis> thaumatin-like protein gene and determination of antimicrobial activity of the in vitro expressed protein

Thaumatin-like proteins (TLPs) are pathogenesis-related proteins, which are involved in plant defense responses to pathogen infection. Expression of the Pinus sylvestris L. TLP gene is up-regulated by methyl jasmonate treatment and inoculation with Heterobasidion annosum. A full-length Pinus taeda TLP gene sequence was used to design PCR primers for amplification of the full-length TLP gene from P. sylvestris. A putative 705-bp open reading frame of TLP gene was cloned into Escherichia coli cells, and then subcloned into the overexpression vector pET100 using BL21 Star expression bacteria. Optimization of the expression of recombinant TLP was achieved by decreasing both expression temperature and IPTG concentration. The purified 24.6-kDa TLP shows antimicrobial activity against 12 fungal species.     

Application of FT-IR spectroscopy for control of the medium composition during the biodegradation of nitro aromatic compounds

Previous studies showed that cabbage leaf extract (CLE) added to the growth medium can noticeably promote the degradation of nitro aromatic compounds by specific consortium of bacteria upon their growth. For further development of the approach for contaminated soil remediation it was necessary to evaluate the qualitative and/or quantitative composition of different origin CLE and their relevance on the growth of explosives-degrading bacteria. Six CLE (different by species, cultivars and harvesting time) were tested and used as additives to the growth medium. It was shown that nitro aromatic compounds can be identified in the FT-IR absorption spectra by the characteristic band at 1,527 cm⁻¹, and in CLE by the characteristic band at 1,602 cm⁻¹. The intensity of the CLE band at 1,602 cm⁻¹ correlated with the concentration of total nitrogen (R ² = 0.87) and decreased upon the growth of bacteria. The content of nitrogen in CLE differed (0.22–1.00 vol.%) and significantly influenced the content of total carbohydrates (9.50–16.00% DW) and lipids [3.90–9.90% dry weight (DW)] accumulated in bacterial cells while the content of proteins was similar in all samples. Though this study showed quantitative differences in the composition of the studied CLE and the response of bacterial cells to the composition of the growth media, and proved the potential of this additive for remediation of contaminated soil. It was shown that analysis of CLE and monitoring of the conversion of nitro aromatic compounds can be investigated by FT-IR spectroscopy as well as by conventional chemical methods.     

Impact of the genes UGT1A1, GSTT1, GSTM1, GSTA1, GSTP1 and NAT2 on acute alcohol-toxic hepatitis

Alcohol metabolism causes cellular damage by changing the redox status of cells. In this study, we investigated the relationship between genetic markers in genes coding for enzymes involved in cellular redox stabilization and their potential role in the clinical outcome of acute alcohol-induced hepatitis. Study subjects comprised 60 patients with acute alcohol-induced hepatitis. The control group consisted of 122 healthy non-related individuals. Eight genetic markers of the genes UGT1A1, GSTA1, GSTP1, NAT2, GSTT1 and GSTM1 were genotyped. GSTT1 null genotype was identified as a risk allele for alcohol-toxic hepatitis progression (OR 2.146, P=0.013). It was also found to correlate negatively with the level of prothrombin (β= ?11.05, P=0.037) and positively with hyaluronic acid (β=170.4, P=0.014). NAT2 gene alleles rs1799929 and rs1799930 showed opposing associations with the activity of the biochemical markers γ-glutamyltransferase and alkaline phosphatase; rs1799929 was negatively correlated with γ-glutamyltransferase (β=?261.3, P=0.018) and alkaline phosphatase (β= ?270.5, P=0.032), whereas rs1799930 was positively correlated with Γ-glutamyltransferase (β=325.8, P=0.011) and alkaline phosphatase (β=374.8, P=0.011). Enzymes of the glutathione S-transferase family and NAT2 enzyme play an important role in the detoxification process in the liver and demonstrate an impact on the clinical outcome of acute alcohol-induced hepatitis.