Autoantibodies against the tumour-associated antigen GA733-2 in patients with colorectal carcinoma

The tumour-associated antigen (TAA) GA733-2 is expressed as a non-secreted surface molecule on the majority of human colorectal carcinoma cells. The antigen has been used as a target for passive and active immunotherapy during the last decade. To determine the incidence of autoantibodies against this antigen, sera from 1068 patients with colorectal carcinoma were analysed for naturally occurring IgG antibodies against the baculovirus-produced GA733-2E protein. A total of 14.5% of the patients had IgG antibodies against the antigen. In 519 patients, sera were collected at the time of diagnosis and 15% of those patients had anti-GA733-2E IgG antibodies. There was a tendency to a higher frequency of patients with antibodies among those in the advanced Dukes stages: 11% in stage A and 32% in stage D respectively (P = 0.06). Antibodies could be detected for up to 10 years after the diagnosis. Patients with Crohn's disease or colitis ulcerosa (n = 20) did not elicit anti-GA733-2E antibodies. No healthy control donor (n = 45) had detectable antibodies against the antigen. The specificity of GA733-2E-reactive serum IgG was indicated by significant inhibition of mAb17-1A (originally used to define GA733-2) binding to the GA733-2E antigen. Sera of positive patients bound to the GA733-2-expressing human colorectal carcinoma cell line, SW948. No significant correlation was found between the presence of antibodies and survival in the present patient population. However, the high incidence of autoantibodies against this tumour antigen in colorectal carcinoma patients confirms its antigenicity in humans and supports the use of the GA733-2 antigen as a target for immunotherapy. Received: 25 May 1998 / Accepted: 26 November 1998     

The epigenetic regulator BRD4 is required for myofibroblast differentiation of knee fibroblasts

Arthrofibrosis, which is characterized by excessive scar tissue and limited motion, can complicate the daily functioning of patients after total knee arthroplasty (TKA). Molecular hallmarks of arthrofibrosis include pathologic accumulation of myofibroblasts and disproportionate collagen deposition. Epigenetic mechanisms, including posttranslation modification of histones, control gene expression and may regulate fibrotic events. This study assessed the role of the bromodomain and extra-terminal (BET) proteins on myofibroblast differentiation. This group of epigenetic regulators recognize acetylated lysines and are targeted by a class of drugs known as BET inhibitors. RNA-seq analysis revealed robust mRNA expression of three BET members (BRD2, BRD3, and BRD4) while the fourth member (BRDT) is not expressed in primary TKA knee outgrowth fibroblasts. RT-qPCR and western blot analyses revealed that BET inhibition with the small molecule JQ1 impairs TGFβ1-induced expression of ACTA2, a key myofibroblast marker, in primary outgrowth knee fibroblasts. Similarly, JQ1 administration also reduced COL3A1 mRNA levels and collagen deposition as monitored by picrosirius red staining. Interestingly, the inhibitory effects of JQ1 on ACTA2 mRNA and protein expression, as well as COL3A1 expression and collagen deposition, were paralleled by siRNA-mediated depletion of BRD4. Together, these data reveal that BRD4-mediated epigenetic events support TGFβ1-mediated myofibroblast differentiation and collagen deposition as seen in arthrofibrosis. To our knowledge, these are the first studies that assess epigenetic regulators and their downstream events in the context of arthrofibrosis. Future studies may reveal clinical utility for drugs that target epigenetic pathways, specifically BET proteins, in the prevention and treatment of arthrofibrosis.     

SELEX and the evolution of genomes

The interrupted genome structures of complex multicellular organisms have most likely changed the evolution of the regulation of metabolism and development. Wasted intron sequences make regulation of gene expression in (for example) mammals appear to be unnecessarily complicated. The recent discoveries that globular RNA molecules are very much like the antigen-combining sites of antibodies suggest that intronic RNA may be used to help solve the problems raised by this complexity.     

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria¹, fungi² and parasites³. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent⁴. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase⁵. Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility⁶.We describe methods to isolate Neutrophil Granulocytes from peripheral human blood⁷ and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.     

Gastric Helicobacter pylori infection is associated with adverse metabolic traits in severely obese subjects

Objective:

Asian studies have reported on an association of Helicobacter pylori (Hp) infection with insulin resistance (IR) in normal‐weight subjects. Whether such an association likewise exists in European subjects with severe obesity was questioned.

Design and Methods:

To address this question, 370 severely obese patients from our database were identified, who had undergone a gastroscopy with a histological examination of gastric mucosal biopsies and a concurrent assessment metabolic blood parameters as a standard examination before bariatric surgery.

Results:

Seventy‐five (20.3%) of the subjects displayed a histologically proven Hp infection (Hp+). Sex distribution, age, and body mass index of Hp+ subjects did not differ from that of the subjects with no Hp infection (Hp–; all P > 0.293), but Hp+ subjects were significantly smaller (P = 0.006). Fasting glucose, insulin, high‐sensitive C reactive protein, and alanine aminotransferase levels as well as calculated indices of IR (i.e., HOMA‐IR, QUICKI) did not differ between Hp+ and Hp– subjects (all P > 0.42). However, Hp+ subjects showed significantly higher triglyceride levels and a higher total chloesterol‐to‐HDL ratio but lower sex‐hormone binding globulin (SHGB) levels than Hp– subjects (all P ≤ 0.05).

Conclusion:

While our results obtained in European severely obese patients do not provide evidence for an enhanced IR state associated with gastric Hp infection, they suggest that the presence of the bacterium in gastric biopsies is associated with an adverse lipid profile and reduced SHGB levels.     

A cross-scale analysis to understand and quantify the effects of photosynthetic enhancement on crop growth and yield across environments

Photosynthetic manipulation provides new opportunities for enhancing crop yield. However, understanding and quantifying the importance of individual and multiple manipulations on the seasonal biomass growth and yield performance of target crops across variable production environments is limited. Using a state-of-the-art cross-scale model in the APSIM platform we predicted the impact of altering photosynthesis on the enzyme-limited (A_c) and electron transport-limited (A_j) rates, seasonal dynamics in canopy photosynthesis, biomass growth, and yield formation via large multiyear-by-location crop growth simulations. A broad list of promising strategies to improve photosynthesis for C₃ wheat and C₄ sorghum were simulated. In the top decile of seasonal outcomes, yield gains were predicted to be modest, ranging between 0% and 8%, depending on the manipulation and crop type. We report how photosynthetic enhancement can affect the timing and severity of water and nitrogen stress on the growing crop, resulting in nonintuitive seasonal crop dynamics and yield outcomes. We predicted that strategies enhancing A_c alone generate more consistent but smaller yield gains across all water and nitrogen environments, A_j enhancement alone generates larger gains but is undesirable in more marginal environments. Large increases in both A_c and A_j generate the highest gains across all environments. Yield outcomes of the tested manipulation strategies were predicted and compared for realistic Australian wheat and sorghum production. This study uniquely unpacks complex cross-scale interactions between photosynthesis and seasonal crop dynamics and improves understanding and quantification of the potential impact of photosynthesis traits (or lack of it) for crop improvement research.     

Treatment of Mycoplasma genitalium. Observations from a Swedish STD Clinic

Objectives

To evaluate therapy for Mycoplasma genitalium infection with doxycycline or azithromycin 1 g compared to five days of azithromycin (total dose 1.5 g).

Methods

A retrospective case study was performed among patients attending the STD-clinic in Falun, Sweden 1998–2005. All patients with a positive PCR test for M. genitalium were routinely offered a test of cure (toc). Response to doxycycline for 9 days, azithromycin 1 g single dose and extended azithromycin (500 mg on day 1 followed by 250 mg o.d. for 4 days) was determined. In patients with treatment failure after azithromycin, macrolide resistance was monitored before and after treatment. Furthermore, the rate of macrolide resistance was monitored for positive specimens available from 2006–2011.

Results

The eradication rate after doxycycline was 43% (48% for women and 38% for men), for azithromycin 1 g 91% (96% for women and 88% for men) and for extended azithromycin 99% (100% for women and 93% for men). Macrolide resistance developed in 7/7 examined (100%) of those testing positive after azithromycin 1 g, but in none of those treated with extended azithromycin. Macrolide resistance before treatment increased from 0% in 2006 and 2007 to 18% in 2011.

Conclusions

These findings confirm the results from other studies showing that doxycycline is inefficient in eradicating M. genitalium. Although azithromycin 1 g was not significantly less efficient than extended dosage, it was associated with selection of macrolide resistant M. genitalium strains and should not be used as first line therapy for M. genitalium. Monitoring of M. genitalium macrolide resistance should be encouraged.     

Arg149 is involved in switching the low affinity, open state of the binding protein AfProX into its high affinity, closed state

The substrate binding protein AfProX from the Archaeoglobus fulgidus ProU ATP binding cassette transporter is highly selective for the compatible solutes glycine betaine (GB) and proline betaine, which confer thermoprotection to this hyperthermophilic archaeon. A detailed mutational analysis of the substrate binding site revealed the contribution of individual amino acids for ligand binding. Replacement of Arg149 by an Ala residue displayed the largest impact on substrate binding. The structure of a mutant AfProX protein (substitution of Tyr111 with Ala) in complex with GB was solved in the open liganded conformation to gain further insight into ligand binding. In this crystal structure, GB is bound differently compared to the GB closed liganded structure of the wild-type AfProX protein. We found that a network of amino acid side chains communicates the presence of GB toward Arg149, which increases ligand affinity and induces domain closure of AfProX. These results were corroborated by molecular dynamics studies and support the view that Arg149 finalizes the high-affinity state of the AfProX substrate binding protein.     

T-cell responses to the DBLα-tag, a short semi-conserved region of the Plasmodium falciparum membrane erythrocyte protein 1

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a variant surface antigen expressed on mature forms of infected erythrocytes. It is considered an important target of naturally acquired immunity. Despite its extreme sequence heterogeneity, variants of PfEMP1 can be stratified into distinct groups. Group A PfEMP1 have been independently associated with low host immunity and severe disease in several studies and are now of potential interest as vaccine candidates. Although antigen-specific antibodies are considered the main effector mechanism in immunity to malaria, the induction of efficient and long-lasting antibody responses requires CD4+ T-cell help. To date, very little is known about CD4+ T-cell responses to PfEMP1 expressed on clinical isolates. The DBLα-tag is a small region from the DBLα-domain of PfEMP1 that can be amplified with universal primers and is accessible in clinical parasite isolates. We identified the dominant expressed PfEMP1 in 41 individual clinical parasite isolates and expressed the corresponding DBLα-tag as recombinant antigen. Individual DBLα-tags were then used to activate CD4+ T-cells from acute and convalescent blood samples in children who were infected with the respective clinical parasite isolate. Here we show that CD4+ T-cell responses to the homologous DBLα-tag were induced in almost all children during acute malaria and maintained in some for 4 months. Children infected with parasites that dominantly expressed group A-like PfEMP1 were more likely to maintain antigen-specific IFNγ-producing CD4+ T-cells than children infected with parasites dominantly expressing other PfEMP1. These results suggest that group A-like PfEMP1 may induce long-lasting effector memory T-cells that might be able to provide rapid help to variant-specific B cells. Furthermore, a number of children induced CD4+ T-cell responses to heterologous DBLα-tags, suggesting that CD4+ T-cells may recognise shared epitopes between several DBLα-tags.