A Characterization of the Oral Microbiome in Allogeneic Stem Cell Transplant Patients

Background

The mouth is a complex biological structure inhabited by diverse bacterial communities. The purpose of this study is to describe the effects of allogeneic stem cell transplantation on the oral microbiota and to examine differences among those patients who acquired respiratory complications after transplantation.

Methodology/Principal Findings

All patients were consented at the National Institutes of Health, Clinical Center. Bacterial DNA was analyzed from patients'' oral specimens using the Human Oral Microbe Identification Microarray. The specimens were collected from four oral sites in 45 allogeneic transplantation patients. Specimens were collected at baseline prior to transplantation, after transplantation at the nadir of the neutrophil count and after myeloid engraftment. If respiratory signs and symptoms developed, additional specimens were obtained. Patients were followed for 100 days post transplantation. Eleven patients'' specimens were subjected to further statistical analysis. Many common bacterial genera, such as Streptococcus, Veillonella, Gemella, Granulicatella and Camplyobacter were identified as being present before and after transplantation. Five of 11 patients developed respiratory complications following transplantation and there was preliminary evidence that the oral microbiome changed in their oral specimens. Cluster analysis and principal component analysis revealed this change in the oral microbiota.

Conclusions/Significance

After allogeneic transplantation, the oral bacterial community''s response to a new immune system was not apparent and many of the most common core oral taxa remained unaffected. However, the oral microbiome was affected in patients who developed respiratory signs and symptoms after transplantation. The association related to the change in the oral microbiota and respiratory complications after transplantation will be validated by future studies using high throughput molecular methods.     

The co-selection of fluoroquinolone resistance genes in the gut flora of vietnamese children

Antimicrobial consumption is one of the major contributing factors facilitating the development and maintenance of bacteria exhibiting antimicrobial resistance. Plasmid-mediated quinolone resistance (PMQR) genes, such as the qnr family, can be horizontally transferred and contribute to reduced susceptibility to fluoroquinolones. We performed an observational study, investigating the copy number of PMQR after antimicrobial therapy. We enrolled 300 children resident in Ho Chi Minh City receiving antimicrobial therapy for acute respiratory tract infections (ARIs). Rectal swabs were taken on enrollment and seven days subsequently, counts for Enterobacteriaceae were performed and qnrA, qnrB and qnrS were quantified by using real-time PCR on metagenomic stool DNA. On enrollment, we found no association between age, gender or location of the participants and the prevalence of qnrA, qnrB or qnrS. Yet, all three loci demonstrated a proportional increase in the number of samples testing positive between day 0 and day 7. Furthermore, qnrB demonstrated a significant increase in copy number between paired samples (p<0.001; Wilcoxon rank-sum), associated with non-fluoroquinolone combination antimicrobial therapy. To our knowledge, this is the first study describing an association between the use of non-fluoroquinolone antimicrobials and the increasing relative prevalence and quantity of qnr genes. Our work outlines a potential mechanism for the selection and maintenance of PMQR genes and predicts a strong effect of co-selection of these resistance determinants through the use of unrelated and potentially unnecessary antimicrobial regimes.     

Results of a psychosomatic training program in China, Vietnam and Laos

ABSTRACT: BACKGROUND: With the "ASIA-LINK" program, the European Community has supported the development and implementation of a curriculum of postgraduate psychosomatic training for medical doctors in China, Vietnam and Laos. Currently, these three countries are undergoing great social, economic and cultural changes. The associated psychosocial stress has led to increases in psychological and psychosomatic problems, as well as disorders for which no adequate medical or psychological care is available, even in cities. Health care in these three countries is characterized by the coexistence of Western medicine and traditional medicine. Psychological and psychosomatic disorders and problems are insufficiently recognized and treated, and there is a need for biopsychosocially orientated medical care. Little is known about the transferability of Western-oriented psychosomatic training programs in the Southeast Asian cultural context. METHODS: The curriculum was developed and implemented in three steps: 1) an experimental phase to build a future teacher group; 2) a joint training program for future teachers and German teachers; and 3) training by Asian trainers that was supervised by German teachers. The didactic elements included live patient interviews, lectures, communication skills training and Balint groups. The training was evaluated using questionnaires for the participants and interviews of the German teachers and the future teachers. RESULTS: Regional training centers were formed In China (Shanghai), Vietnam (Ho Chi Minh City and Hue) and Laos (Vientiane). A total of 200 physicians completed the training, and 30 physicians acquired the status of future teacher. The acceptance of the training was high, and feelings of competence increased during the courses. The interactive training methods were greatly appreciated, with the skills training and self-experience ranked as the most important topics. Adaptations to the cultural background of the participants were necessary for the topics of "breaking bad news," the handling of negative emotions, discontinuities in participation, the hierarchical doctor-patient relationship, culture-specific syndromes and language barriers. In addition to practical skills for daily clinical practice, the participants wanted to learn more about didactic teaching methods. Half a year after the completion of the training program, the participants stated that the program had a great impact on their daily medical practice. CONCLUSIONS: The training in psychosomatic medicine for postgraduate medical doctors resulted in a positive response and is an important step in addressing the barriers in providing psychosomatic primary care. The transferability of western concepts should be tested locally, and adaptations should be undertaken where necessary. The revised curriculum forms the basis of training in psychosomatic medicine and psychotherapy for medical students and postgraduate doctors in China, Vietnam and Laos.     

Hyperosmotic stress induces Axl activation and cleavage in cerebral endothelial cells

Because of the relative impermeability of the blood‐brain barrier (BBB), many drugs are unable to reach the CNS in therapeutically relevant concentration. One method to deliver drugs to the CNS is the osmotic opening of the BBB using mannitol. Hyperosmotic mannitol induces a strong phosphorylation on tyrosine residues in a broad spectrum of proteins in cerebral endothelial cells, the principal components of the BBB. Previously, we have shown that among targets of tyrosine phosphorylation are β‐catenin, extracellular signal‐regulated kinase 1/2 and the non‐receptor tyrosine kinase Src. The aim of this study was to identify new signalling pathways activated by hypertonicity in cerebral endothelial cells. Using an antibody array and immunoprecipitation we identified the receptor tyrosine kinase Axl to become tyrosine phosphorylated in response to hyperosmotic mannitol. Besides activation, Axl was also cleaved in response to osmotic stress. Degradation of Axl proved to be metalloproteinase‐ and proteasome‐dependent and resulted in 50–55 kDa C‐terminal products which remained phosphorylated even after degradation. Specific knockdown of Axl increased the rate of apoptosis in hyperosmotic mannitol‐treated cells; therefore, we assume that activation of Axl may be a protective mechanism against hypertonicity‐induced apoptosis. Our results identify Axl as an important element of osmotic stress‐induced signalling.     

An accurate definition of the status of inactive hepatitis B virus carrier by a combination of biomarkers (FibroTest-ActiTest) and viral load

Background

The combination of transaminases (ALT), biopsy, HBeAg and viral load have classically defined the inactive status of carriers of chronic hepatitis B. The use of FibroTest (FT) and ActiTest (AT), biomarkers of fibrosis and necroinflammatory activity, has been previously validated as alternatives to biopsy. We compared the 4-year prognostic value of combining FT-AT and viral load for a better definition of the inactive carrier status.

Methods and Findings

1,300 consecutive CHB patients who had been prospectively followed since 2001 were pre-included. The main endpoint was the absence of liver-related complications, transplantation or death. We used the manufacturers'' definitions of normal FT (< = 0.27), normal AT (< = 0.29) and 3 standard classes for viral load. The adjustment factors were age, sex, HBeAg, ethnic origin, alcohol consumption, HIV-Delta-HCV co-infections and treatment.

Results

1,074 patients with baseline FT-AT and viral load were included: 41 years old, 47% African, 27% Asian, 26% Caucasian. At 4 years follow-up, 50 complications occurred (survival without complications 93.4%), 36 deaths occurred (survival 95.0%), including 27 related to HBV (survival 96.1%). The prognostic value of FT was higher than those of viral load or ALT when compared using area under the ROC curves [0.89 (95%CI 0.84–0.93) vs 0.64 (0.55–0.71) vs 0.53 (0.46–0.60) all P<0.001], survival curves and multivariate Cox model [regression coefficient 5.2 (3.5–6.9; P<0.001) vs 0.53 (0.15–0.92; P = 0.007) vs −0.001 (−0.003−0.000;P = 0.052)] respectively. A new definition of inactive carriers was proposed with an algorithm combining “zero” scores for FT-AT (F0 and A0) and viral load classes. This new algorithm provides a 100% negative predictive value for the prediction of liver related complications or death. Among the 275 patients with the classic definition of inactive carrier, 62 (23%) had fibrosis presumed with FT, and 3 died or had complications at 4 year.

Conclusion

In patients with chronic hepatitis B, a combination of FibroTest-ActiTest and viral load testing accurately defined the prognosis and the inactive carrier status.     

Identification of lipopolysaccharide binding site on high molecular weight kininogen

Plasma kallikrein kinin system (KKS) activation along with its cellular receptors expression are increased after injury and in patients with septic shock, hypotensive bacteremia and rhesus monkey infected with Salmonella typhimurium. KKS signaling cascade is activated by activated factor XII (FXIIa, Hageman factor)- and prolylcarboxypeptidase (PRCP)-dependent pathways on endothelial cells. Among the many entities that comprise the KKS, high molecular weight kininogen (HK), a bradykinin precursor, is critical in the assembly and activation of this system. HK is primarily expressed in the liver and secreted into the bloodstream. The activation of the KKS influences the permeability of the endothelium by liberating bradykinin (BK) from HK. BK is a potent inflammatory peptide which stimulates constitutive bradykinin B2 and inducible B1 receptors to release nitric oxide and prostacyclin. Regardless of the triggers, PK can only be activated on HK bound to the artificial negatively charged or to cell membrane surfaces. Since LPS has a negatively charged moiety and the ability to induce inflammatory responses in human, we determined the interaction between LPS and HK. HKH19 (HK cell binding site) and heparin inhibited LPS binding to HK with IC₅₀s of 15 nM and 20 μg/ml, respectively. C1-inhibitor and N-acetylglucosamine glycan inhibited LPS binding to HK with IC₅₀s of about 10 μg/ml and 10 mM, respectively. This novel study underscores the implication of HK in infection. We propose that HKH19, heparin, and C1-inhibitor present therapeutic potential for the treatment of sepsis and hypotensive bacteremia.     

Computational complexity of a problem in molecular structure prediction.

The computational task of protein structure prediction is believed to require exponential time, but previous arguments as to its intractability have taken into account only the size of a protein's conformational space. Such arguments do not rule out the possible existence of an algorithm, more selective than exhaustive search, that is efficient and exact. (An efficient algorithm is one that is guaranteed, for all possible inputs, to run in time bounded by a function polynomial in the problem size. An intractable problem is one for which no efficient algorithm exists.) Questions regarding the possible intractability of problems are often best answered using the theory of NP-completeness. In this treatment we show the NP-hardness of two typical mathematical statements of empirical potential energy function minimization of macromolecules. Unless all NP-complete problems can be solved efficiently, these results imply that a function minimization algorithm can be efficient for protein structure prediction only if it exploits protein-specific properties that prohibit the simple geometric constructions that we use in our proofs. Analysis of further mathematical statements of molecular structure prediction could constitute a systematic methodology for identifying sources of complexity in protein folding, and for guiding development of predictive algorithms.     

DEVELOPMENTAL CHANGES IN THE KINETICS OF γ-AMINOBUTYRIC ACID TRANSPORT BY CHICK RETINA

—Isolated retinas from chick embryos and mature animals were incubated in [³H]GABA at 25°C for 10 min in order to investigate kinetic properties of the amino acid uptake system. Embryo retina accumulated [³H]GABA by two distinct kinetic systems with K_m values of the order 10⁻⁴m and 10⁻⁵m for the low- and high-affinity mechanisms respectively. However, as the retina matured, the high-affinity process disappeared and only the low-affinity system was detectable. No obvious explanation can be offered for this phenomenon although a similar observation has previously been made in chick brain by other workers.