Effect of Plant Systemic Resistance Elicited by Biological and Chemical Inducers on the Colonization of the Lettuce and Basil Leaf Apoplast by Salmonella enterica.

Mitigation strategies to prevent microbial contamination of crops are lacking. We tested the hypothesis that induction of plant systemic resistance by biological (induced systemic resistance [ISR]) and chemical (systemic acquired resistance [SAR]) elicitors reduces endophytic colonization of leaves by Salmonella enterica serovars Senftenberg and Typhimurium. S. Senftenberg had greater endophytic fitness than S. Typhimurium in basil and lettuce. The apoplastic population sizes of serovars Senftenberg and Typhimurium in basil and lettuce, respectively, were significantly reduced approximately 10- to 100-fold by root treatment with microbial inducers of systemic resistance compared to H2O treatment. Rhodotorula glutinis effected the lowest population increases of S. Typhimurium in lettuce and S. Senftenberg in basil leaves, respectively 120- and 60-fold lower than those seen with the H2O treatment over 10 days postinoculation. Trichoderma harzianum and Pichia guilliermondii did not have any significant effect on S. Senftenberg in the basil apoplast. The chemical elicitors acidobenzolar-S-methyl and dl-ß-amino-butyric acid inhibited S. Typhimurium multiplication in the lettuce apoplast 10- and 2-fold, respectively, compared to H2O-treated plants. All ISR and SAR inducers applied to lettuce roots in this study increased leaf expression of the defense gene PR1, as did Salmonella apoplastic colonization in H2O-treated lettuce plants. Remarkably, both acidobenzolar-S-methyl upregulation and R. glutinis upregulation of PR1 were repressed by the presence of Salmonella in the leaves. However, enhanced PR1 expression was sustained longer and at greater levels upon elicitor treatment than by Salmonella induction alone. These results serve as a proof of concept that priming of plant immunity may provide an intrinsic hurdle against the endophytic establishment of enteric pathogens in leafy vegetables. IMPORTANCE Fruit and vegetables consumed raw have become an important vehicle of foodborne illness despite a continuous effort to improve their microbial safety. Salmonella enterica has caused numerous recalls and outbreaks of infection associated with contaminated leafy vegetables. Evidence is increasing that enteric pathogens can reach the leaf apoplast, where they confront plant innate immunity. Plants may be triggered for induction of their defense signaling pathways by exposure to chemical or microbial elicitors. This priming for recognition of microbes by plant defense pathways has been used to inhibit plant pathogens and limit disease. Given that current mitigation strategies are insufficient in preventing microbial contamination of produce and associated outbreaks, we investigated the effect of plant-induced resistance on S. enterica colonization of the lettuce and basil leaf apoplast in order to gain a proof of concept for the use of such an intrinsic approach to inhibit human pathogens in leafy vegetables.

Mass gathering events and undetected transmission of SARS-CoV-2 in vulnerable populations leading to an outbreak with high case fatality ratio in the district of Tirschenreuth, Germany.

In early March 2020, a COVID-19-outbreak occurred in the district of Tirschenreuth, Germany. The outbreak was characterised by a rapid increase in case numbers and a comparatively high crude case fatality ratio (CFR; 11%). Until the beginning of May 2020, 1122 cases were reported in the district. To investigate the outbreak, we analysed surveillance and other data available at the district health department, including data on cases living in care facilities and public health measures applied. Furthermore, we compared the number of tests performed in Tirschenreuth and in Germany as a whole. We interviewed the first 110 cases in order to investigate potential exposures at the beginning of the outbreak. We found that returning ski-travellers from Austria and Italy and early undetected community transmission likely initiated the outbreak which was then accelerated by Bavarian beer festivities. Testing of mainly acute cases in the district of Tirschenreuth resulted in a higher rate of positive tests compared to the whole of Germany. Despite adjustment for age, the CFR continued to exceed the German mean which was due to spread to vulnerable populations. Strict public health measures likely contributed to control the outbreak by mid-April 2020.

Are men who have sex with men in Europe protected from hepatitis B?

Hepatitis B vaccination is recommended for men who have sex with men (MSM) in many countries, but information on vaccine coverage is scarce. We studied hepatitis B vaccination programmes and coverage among MSM in Europe to guide prevention. From a large (N = 174 209) pan-European MSM survey (EMIS-2010), we used data on self-reported hepatitis B vaccination, age, education, settlement size and disclosure of the same-sex sexual orientation ('outness'). We excluded participants with a history of hepatitis B. In multilevel (participants, countries) logistic regression models, we calculated adjusted odds ratios (aOR) with 95% confidence intervals (95% CI). We analysed data of 163 987 MSM in 38 European countries: 38.3% were 'out' to all or almost all, 56.4% reported vaccination against hepatitis B and 65.5% lived in countries with free recommended hepatitis B vaccination for MSM. In the final model the odds for being vaccinated increased with outness ('out to all or almost all': aOR 1.76, 95% CI 1.70-1.83 vs. 'out to no one') and with living in countries, where hepatitis B vaccination was recommended and free-of-charge for MSM (aOR 2.21, 95% CI 1.47-3.32 vs. 'no or unclear recommendation'). To increase hepatitis B vaccination coverage among MSM, implementation of MSM-specific recommendations and improvement of the societal climate for MSM is needed.

Efficacy and safety of whole body vibration in maintenance hemodialysis patients - A pilot study.

OBJECTIVES: The aim of this study was to assess safety and effectiveness of Whole Body Vibration exercise (WBV) to improve physical performance and parameters of inflammation in patients on maintenance hemodialysis (MHD). METHODS: Prospective, open-label trial in n=14 patients on maintenance hemodialysis. Participants performed WBV twice weekly for 12 weeks before (n=8) or after (n=6) hemodialysis sessions. The primary endpoint was physical performance assessed by the Short-Physical-Performance-Battery (SPPB). Secondary endpoints included established parameters of musculoskeletal assessment and blood chemistry. RESULTS: As compared to baseline, physical performance (SPPB) improved significantly (p=0.035). Moderate advances were also seen for 6-Minute-Walking test, Timed-up-and-go test, jumping height and handgrip strength. Improvements were particularly pronounced in subjects with seriously impaired baseline performance. Skeletal muscle index showed a tendency to better values. Laboratory data exhibited a significant reduction of white blood cell count and a trend to lower levels of hsCRP. WBV was generally well tolerated. Two events of clinically significant blood pressure decline occurred in patients exercising after dialysis sessions. CONCLUSIONS: Results of this pilot study suggest effectiveness and safety of WBV in hemodialysis patients. Beneficial effects may affect both, parameters of physical performance and systemic inflammatory activity, which should be verified in larger scale clinical trials.

The Polymorphic Aggregative Phenotype of Shiga Toxin-Producing Escherichia coli O111 Depends on RpoS and Curli.

Escherichia coli O111 is an emerging non-O157:H7 serotype of Shiga toxin-producing E. coli (STEC). We previously reported that outbreak and environmental, but not sporadic-case, strains of STEC O111 share a distinct aggregation phenotype (M. E. Diodati, A. H. Bates, M. B. Cooley, S. Walker, R. E. Mandrell, and M. T. Brandl, Foodborne Pathog Dis 12:235-243, 2015, http://dx.doi.org/10.1089/fpd.2014.1887). We show here the natural occurrence of nonaggregative variants in single STEC O111 strains. These variants do not produce curli fimbriae and lack RpoS function but synthesize cellulose. The deletion of csgBAC or rpoS in an aggregative outbreak strain abolished aggregate formation, which was rescued when curli biogenesis or RpoS function, respectively, was restored. Complementation of a nonaggregative variant with RpoS also conferred curli production and aggregation. These observations were supported by Western blotting with an anti-CsgA antibody. Immunomicroscopy revealed that curli were undetectable on the cells of the nonaggregative variant and the RpoS mutant but were present in large quantities in the intercellular matrix of the assemblages formed by aggregative strains. Sequence analysis of rpoS in the aggregative strain and its variant showed a single substitution of threonine for asparagine at amino acid 124. Our results indicate that the multicellular behavior of STEC O111 is RpoS dependent via positive regulation of curli production. Aggregation may confer a fitness advantage in O111 outbreak strains under stressful conditions in hydrodynamic environments along the food production chain and in the host, while the occurrence of nonaggregative variants may allow the cell population to adapt to conditions benefiting a planktonic lifestyle.

Pre-clinical endodontic training with artificial instead of extracted human teeth: does the type of exercise have an influence on clinical endodontic outcomes?

AIM: To evaluate whether artificial resin teeth could replace extracted human teeth in pre-clinical endodontic training and if this teaching approach influences the outcome of root canal treatment on patients. METHODOLOGY: In a pre-clinical training course, students of group 1 (n = 44) performed simulated endodontic exercises on four plastic blocks and three extracted human teeth. Students of group 2 (n = 45) performed their exercises on plastic blocks and artificial resin teeth (Real-T Endo, Acadental, Lenexa, KS, USA). Both groups performed their first root canal treatments on patients in the following term. Radiographs taken during root canal treatment were used for the evaluation of treatment outcome. Distances between the master cone or the root filling and the radiographic apex as well as iatrogenic errors were assessed, and comparisons were made using Fischer's exact test. RESULTS: In the pre-clinical course root canal treatments performed by students of group 2 were more often classified as acceptable and a higher number of iatrogenic errors were observed in group 1. When root canal treatments were performed on patients for the first time, no significant difference was observed between the groups in terms of radiographic technical quality of root fillings. CONCLUSIONS: The application of artificial teeth instead of extracted human teeth had no effect on the technical quality of root fillings in terms of position in relation to the root apex or the creation of aberrations.

Identification of Salmonella enterica genes with a role in persistence on lettuce leaves during cold storage by recombinase-based in vivo expression technology.

Recurrent outbreaks of enteric illness linked to lettuce and a lack of efficacious strategies to decontaminate produce underscores the need for a better understanding of the molecular interactions of foodborne pathogens with plants. This study aimed at identifying Salmonella enterica genes involved in the persistence of this organism on post-harvest lettuce during cold storage using recombinase-based in vivo expression technology (RIVET). In total, 37 potentially induced loci were identified in four distinct screenings. Knockout mutations in eight upregulated genes revealed that four of them have a role in persistence of the pathogen in this system. These genes included stfC, bcsA, misL, and yidR, encoding a fimbrial outer membrane usher, a cellulose synthase catalytic subunit, an adhesin of the autotransporter family expressed from the Salmonella pathogenicity island-3, and a putative ATP/GTP-binding protein, respectively. bcsA, misL, and yidR but not stfC mutants were impaired also in attachment and biofilm formation, suggesting that these functions are required for survival of S. enterica on post-harvest lettuce. This is the first report that MisL, which has a role in Salmonella binding to fibronectin in animal hosts, is involved also in adhesion to plant tissue. Hence, our study uncovered a new plant attachment factor in Salmonella and demonstrates that RIVET is an effective approach for investigating human pathogen-plant interactions in a post-harvest leafy vegetable.

Epidemiology of healthcare-associated SARS-CoV-2 outbreaks in Germany between March 2020 and May 2022.

BACKGROUND: Outbreaks in healthcare facilities played a pivotal role in the course of the coronavirus (COVID-19) pandemic. AIM: To investigate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreaks in hospitals, outpatient care, and rehabilitation facilities in Germany from March 2020 to May 2022. METHODS: Data from the German mandatory notification system were used to describe outbreaks by number of cases and case fatality ratio (CFR), and outbreak cases by age and gender. Using Pearson correlation, the dynamics of cases in the general population were compared with cases in healthcare-associated infection (HAI) SARS-CoV-2 outbreaks before and after the start of the vaccination campaign. Additionally, a counterfactual scenario was used to estimate numbers of prevented HAI cases, using the phase before vaccination as baseline. FINDINGS: By the end of May 2022, 8941 healthcare-associated outbreaks were observed with 73,626 cases: 51,504 in hospitals, 15,524 in outpatient care, and 6598 in rehabilitation facilities. Median number of cases per outbreak was 4 (range: 2-342) and cases were more frequently reported in women with 46,818 (63.6%). Overall CFR was 8.1%, higher in men (12.4%) than in women (5.7%). After the vaccination campaign was fully introduced, the association between increasing incidence in the general population and consecutive outbreak cases was decreased by a factor of 10. Furthermore, our counterfactual analysis suggests that more than 55,000 outbreak cases could have been prevented until the end of 2021. CONCLUSION: The vaccination campaign in combination with non-pharmaceutical measures was key to reduce number, size and CFR of healthcare-associated outbreaks.

A target-orientated algorithm for regional citrate-calcium anticoagulation in extracorporeal therapies.

BACKGROUND: Citrate anticoagulation offers several advantages in comparison to conventional anticoagulation. Most algorithms for regional citrate-calcium anticoagulation are based on citrate and calcium chloride infusion coupled in a fixed proportion to the blood flow without considering the hematocrit (Hct)/plasma flow or the filter clearance of citrate and calcium. METHODS: The aim of this study was to develop an algorithm for optimized citrate anticoagulation in extracorporeal therapies such as dialysis. A mathematical model was developed to calculate the volume of citrate infusion required to achieve a desired ionized calcium (iCa) target level in the extracorporeal circuit and to restore the total calcium level to a physiological value. RESULTS: The model was validated by correlation analyses for different blood Hct values and shows an excellent fit to the laboratory measurements. CONCLUSION: The results for both iCa target concentrations, namely those after citrate and calcium infusion, proved that the software algorithm adapts well to variable treatment parameters.

Physicochemical properties of lipid nanoparticles: effect of lipid and surfactant composition.

Understanding the effect of lipid and surfactant composition on particle size and colloidal stability plays a pivotal role in designing lipid nanoparticles (LN) for drug delivery. With respect to our long-term goal, LN for brain delivery, formulations containing lipids and surfactants suitable for intravenous (i.v.) application were selected for the current formulation screening study. LN were prepared by hot high pressure homogenization (HPH) and were characterized during 1 year in terms of macroscopic appearance, particle size by photon correlation spectroscopy (PCS) and optical single particle sizing (OSPS), zeta potential (ZP), as well as physical state and polymorphism by differential scanning calorimetry (DSC). The LN dispersions showed a wide variability in macroscopic appearance, mean size and colloidal stability. Influence factors were the type and concentration of both, the lipid and surfactant component used. The most promising LN showed a small mean size (< 200 nm), a low polydispersity index (PI), (< 0.25) absence of particles in the several-micron range, and a slightly negative ZP (> -12 mV); DSC revealed that some represented supercooled liquids; such LN may be stable at room temperature for at least 1 year. The obtained results are regarded helpful for defining the design space for LN delivery systems, i.e., identifying possible designs and design parameters within the given HPH technology to be applied during future formulation development studies.

Interactions of Salmonella enterica with lettuce leaves.

AIMS: To investigate the interactions of Salmonella enterica with abiotic and plant surfaces and their effect on the tolerance of the pathogen to various stressors. METHODS AND RESULTS: Salmonella strains were tested for their ability to form biofilm in various growth media using a polystyrene plate model. Strong biofilm producers were found to attach better to intact Romaine lettuce leaf tissue compared to weak producers. Confocal microscopy and viable count studies revealed preferential attachment of Salmonella to cut-regions of the leaf after 2 h at 25 degrees C, but not for 18 h at 4 degrees C. Storage of intact lettuce pieces contaminated with Salmonella for 9 days at 4 degrees C resulted only in small changes in population size. Exposure of lettuce-associated Salmonella cells to acidic conditions (pH 3.0) revealed increased tolerance of the attached vs planktonic bacteria. CONCLUSIONS: Biofilm formation on polystyrene may provide a suitable model to predict the initial interaction of Salmonella with cut Romaine lettuce leaves. Association of the pathogen with lettuce leaves facilitates its persistence during storage and enhances its acid tolerance. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the interactions between foodborne pathogens and lettuce might be useful in developing new approaches to prevent fresh produce-associated outbreaks.

More hydrogen bonds for the (structural) biologist.

Why does a given protein structure form and why is this structure stable? These fundamental biochemical questions remain fascinating and challenging problems because the physical bases of the forces that govern protein structure, stability and folding are still not well understood. Now, a general concept of hydrogen bonding in proteins is emerging. This concept involves not only N-H and O-H donor groups, but also C-H, and not only N and O as acceptor groups, but also pi-systems. We postulate that the incorporation of the entirety of these interactions leads to a more complete description of the problem, and that this could provide new perspectives and possibly new answers.

Plant lesions promote the rapid multiplication of Escherichia coli O157:H7 on postharvest lettuce.

Several outbreaks of Escherichia coli O157:H7 infections have been associated with minimally processed leafy vegetables in the United States. Harvesting and processing cause plant tissue damage. In order to assess the role of plant tissue damage in the contamination of leafy greens with E. coli O157:H7, the effect of mechanical, physiological, and plant disease-induced lesions on the growth of this pathogen on postharvest romaine lettuce was investigated. Within only 4 h after inoculation, the population sizes of E. coli O157:H7 increased 4.0-, 4.5-, and 11.0-fold on lettuce leaves that were mechanically bruised, cut into large pieces, and shredded into multiple pieces, respectively. During the same time, E. coli O157:H7 population sizes increased only twofold on leaves that were left intact after harvest. Also, the population size of E. coli O157:H7 was 27 times greater on young leaves affected by soft rot due to infection by Erwinia chrysanthemi than on healthy middle-aged leaves. Confocal microscopy revealed that leaf tip burn lesions, which are caused by a common physiological disorder of lettuce, harbored dense populations of E. coli O157:H7 cells both internally and externally. Investigation of the colonization of cut lettuce stems by E. coli O157:H7 showed that the pathogen grew 11-fold over 4 h of incubation after its inoculation onto the stems, from which large amounts of latex were released. The results of this study indicate that plant tissue damage of various types can promote significant multiplication of E. coli O157:H7 over a short time and suggest that harvesting and processing are critical control points in the prevention or reduction of E. coli O157:H7 contamination of lettuce.

Leaf age as a risk factor in contamination of lettuce with Escherichia coli O157:H7 and Salmonella enterica.

Outbreaks of Escherichia coli O157:H7 infections have been linked increasingly to leafy greens, particularly to lettuce. We present here the first evidence that this enteric pathogen can multiply on the leaves of romaine lettuce plants. The increases in population size of E. coli O157:H7 in the phyllosphere of young lettuce plants ranged from 16- to 100-fold under conditions of warm temperature and the presence of free water on the leaves and varied significantly with leaf age. The population size was consistently ca. 10-fold higher on the young (inner) leaves than on the middle leaves. The growth rates of Salmonella enterica and of the natural bacterial microflora were similarly leaf age dependent. Both enteric pathogens also achieved higher population sizes on young leaves than on middle leaves harvested from mature lettuce heads, suggesting that leaf age affects preharvest as well as postharvest colonization. Elemental analysis of the exudates collected from the surfaces of leaves of different ages revealed that young-leaf exudates were 2.9 and 1.5 times richer in total nitrogen and carbon, respectively, than middle-leaf exudates. This trend mirrored the nitrogen and carbon content of the leaf tissue. Application of ammonium nitrate, but not glucose, to middle leaves enhanced the growth of E. coli O157:H7 significantly, suggesting that low nitrogen limits its growth on these leaves. Our results indicate that leaf age and nitrogen content contribute to shaping the bacterial communities of preharvest and postharvest lettuce and that young lettuce leaves may be associated with a greater risk of contamination with E. coli O157:H7.

Gallium-rich Pd-Ga phases as supported liquid metal catalysts.

A strategy to develop improved catalysts is to create systems that merge the advantages of heterogeneous and molecular catalysis. One such system involves supported liquid-phase catalysts, which feature a molecularly defined, catalytically active liquid film/droplet layer adsorbed on a porous solid support. In the past decade, this concept has also been extended to supported ionic liquid-phase catalysts. Here we develop this idea further and describe supported catalytically active liquid metal solutions (SCALMS). We report a liquid mixture of gallium and palladium deposited on porous glass that forms an active catalyst for alkane dehydrogenation that is resistant to coke formation and is thus highly stable. X-ray diffraction and X-ray photoelectron spectroscopy, supported by theoretical calculations, confirm the liquid state of the catalytic phase under the reaction conditions. Unlike traditional heterogeneous catalysts, the supported liquid metal reported here is highly dynamic and catalysis does not proceed at the surface of the metal nanoparticles, but presumably at homogeneously distributed metal atoms at the surface of a liquid metallic phase.

Cryogenic setup for trapped ion quantum computing.

We report on the design of a cryogenic setup for trapped ion quantum computing containing a segmented surface electrode trap. The heat shield of our cryostat is designed to attenuate alternating magnetic field noise, resulting in 120 dB reduction of 50 Hz noise along the magnetic field axis. We combine this efficient magnetic shielding with high optical access required for single ion addressing as well as for efficient state detection by placing two lenses each with numerical aperture 0.23 inside the inner heat shield. The cryostat design incorporates vibration isolation to avoid decoherence of optical qubits due to the motion of the cryostat. We measure vibrations of the cryostat of less than ±20 nm over 2 s. In addition to the cryogenic apparatus, we describe the setup required for an operation with 40Ca+ and 88Sr+ ions. The instability of the laser manipulating the optical qubits in 40Ca+ is characterized by yielding a minimum of its Allan deviation of 2.4 ⋅ 10-15 at 0.33 s. To evaluate the performance of the apparatus, we trapped 40Ca+ ions, obtaining a heating rate of 2.14(16) phonons/s and a Gaussian decay of the Ramsey contrast with a 1/e-time of 18.2(8) ms.

Entrapment of haemoglobin into liposomes by the dehydration-rehydration method: vesicle characterization and in vivo behaviour.

Haemoglobin (Hb) was isolated from human erythrocytes under conditions which maintained NADH-cytochrome-b5 reductase activity and suppressed oxidation of Hb during storage at 4 degrees C (methaemoglobin values < 3% after 29 days). Hb was entrapped into liposomes composed of hydrogenated egg phosphatidylcholine and equimolar cholesterol according to the dehydration/rehydration procedure of Kirby and Gregoriadis ((1984) Biotechnology 2, 979). However, encapsulation of Hb in its intact form was poor (< 5%) as a result of its oxidation and denaturation during freeze-drying. The addition of cryoprotectants and the use of both, higher initial concentrations of Hb and very small void vesicles resulted in Hb-rich dehydration/rehydration vesicles (phospholipid/Hb molar ratio of about 200:1) of the preferred size of 110 nm (mean). Highly homogeneous and small void vesicles as starting material were prepared using the one-step method of Brandl et al. ((1990) Drug Dev. Ind. Pharm. 16, 2167). The cryoprotectants were chosen with respect to their sufficient protection of Hb without affecting its loading into vesicles during freeze-drying and rehydration. 51Cr-labelling of Hb was used for the in vivo monitoring of the fate of Hb-containing vesicles rather than 125I-labelling since the latter induced strong interactions of Hb with liposomes. Upon intravenous administration into rats, liposomal 51Cr-Hb showed greater blood levels and prolonged circulation times in the blood compared to free Hb. The present approach provides high yield entrapment of labile molecules into vesicles of small size known to exhibit long circulation time.

Molecular dynamics simulation reveals conformational switching of water-mediated uracil-cytosine base-pairs in an RNA duplex.

A 4 ns molecular dynamics simulation of an RNA duplex (r-GGACUUCGGUCC)(2 )in solution with Na+ and Cl- as counterions was performed. The X-ray structure of this duplex includes two water-mediated uracil-cytosine pairs. In contrast to the other base-pairs in the duplex the water-mediated pairs switch between different conformations. One conformation corresponds to the geometry of the water-mediated UC pairs in the duplex X-ray structure with water acting both as hydrogen-bond donor and acceptor. Another conformation is close to that of a water-mediated UC base-pair found in the X-ray structure of the 23 S rRNA sarcin/ricin domain. In this case the oxygen of the water molecule is linked to two-base donor sites. For a very short time also a direct UC base-pair and a further conformation that is similar to the one found in the RNA duplex structure but exhibits an increased H3(U)...N3(C) distance is observed. Water molecules with unusually long residence times are involved in the water-mediated conformations. These results indicate that the dynamic behaviour of the water-mediated UC base-pairs differs from that of the duplex Watson-Crick and non-canonical guanine-uracil pairs with two or three direct hydrogen bonds. The conformational variability and increased flexibility has to be taken into account when considering these base-pairs as RNA building blocks and as recognition motifs.

C-H...pi-interactions in proteins.

A non-redundant set of 1154 protein structures from the Protein Data Bank was examined with respect to close interactions between C-H-donor and pi-acceptor groups. A total of 31,087 interactions were found to satisfy our selection criteria. Their geometric parameters suggest that these interactions can be classified as weak hydrogen bonds.A set of 12 interaction classes were defined based on the division of the donors into three groups and the acceptors into four groups. These classes were examined separately, and the respective interactions described in detail in each class. Most prominent were interactions between aliphatic C-H donors and aromatic pi-acceptors and interactions between aromatic C-H donors and aromatic pi-acceptors. About three-quarters of the Trp-rings, half of all Phe and Tyr-rings and a quarter of all His-rings were found to be involved as acceptors in C-H...pi-interactions. On the donor side, a preference for aromatic C-H groups was observed, but also for the aliphatic side-chains of the long, extended amino acid residues Lys, Arg and Met, and the Pro ring. The average distance between the C-donor and the center-of-mass of the pi-acceptor was observed to be significantly longer in the 174 protein structures determined at >2.5 A resolution. Also, the distribution is significantly wider. This resolution dependence suggests that the force fields commonly used for the refinement of protein structures may not be adequate. C-H...pi-interactions involving aromatic groups either as donor or as acceptor groups are found mostly in the interior of the protein. The more hydrophilic the participating groups are, the closer to the surface are the interactions located. About 40 % of all C-H...pi-interactions occur between amino acid residue side-chains that are separated by nine or less residues in sequence. Dependent on the interaction class, different preferences for secondary structure, residue type and side-chain conformation were observed. It is likely that the C-H...pi-interactions contribute significantly to the overall stability of a protein.

Bispecific antibody fragments with CD20 X CD28 specificity allow effective autologous and allogeneic T-cell activation against malignant cells in peripheral blood and bone marrow cultures from patients with B-cell lineage leukemia and lymphoma.

Bispecific antibodies directed against tumor-associated target antigens and to surface receptors mediating T-cell activation, such as the TCR/CD3 complex and the costimulatory receptor CD28, are capable of mediating T-cell activation resulting in tumor cell killing. In this study, we used the B-cell-associated antigens CD19 and CD20 as target structures on human leukemic cells. We found that a combination of bispecific antibody fragments (bsFab2) with target x CD3 and target x CD28 specificity induces vigorous autologous T-cell activation and killing of malignant cells in peripheral blood and bone marrow cultures from patients with chronic lymphocytic leukemia and follicular lymphoma. The bsFab2 targeting CD20 were considerably more effective than those binding to CD19. The colony-forming capacity of treated bone marrow was impaired due to large amounts of tumor necrosis factor alpha produced during bsFab2-induced T-cell activation. Neutralizing tumor necrosis factor alpha antibodies were found to reverse this negative effect without affecting T-cell activation and tumor cell killing. CD20 x CD28 bsFab2, when used alone rather than in combination, markedly improved the recognition of leukemic cells by allogeneic T cells. Therefore, these reagents may be capable of enhancing the immunogenicity of leukemic cells in general and, in particular, of increasing the antileukemic activity of allogeneic donor buffy coat cells in relapsed bone marrow transplanted patients.