Sterilization efficacy of a new water-free breast milk pasteurizer.

BACKGROUND: Breast milk, nature's optimum source of nutrition for infants, can contain undesirable microorganisms that cause severe morbidity. After an outbreak of multidrug-resistant Escherichia coli among neonates receiving breast milk donated by another mother in our neonatal intensive care unit (NICU), we were motivated to develop a high-grade breast milk pasteurizer (BMP) designed to thaw and pasteurize breast milk at 63°C for 30 min in a sealed bag without having to open the bag or immerse it in water. METHODS: Pre-existing bacteria and spiked cytomegalovirus (CMV) were measured pre- and post-pasteurization in frozen breast milk donated by mothers of children admitted to the NICU. RESULTS: Among 48 breast milk samples (mean ± standard deviation [SD]), pre-existing bacterial counts of 5.1±1.1 × 104 colony forming units (cfu)/mL decreased to less than 10 cfu/mL (below detection level) in 45 samples after pasteurization for 30 min. In three samples, 10-110 cfu/mL persisted. As no CMV was detected in any of the 48 samples, CMV at ≥5 × 104 pfu/mL was spiked into 11 breast milk samples. After just 10 min of pasteurization, infectious CMV was not detected (threshold <50 pfu/mL) in any sample. CONCLUSION: A new BMP was shown to pasteurize milk effectively with more than a 3-log reduction of microorganisms. Compared to conventional pasteurizers, this device reduces the effort involved in pasteurizing breast milk, avoids various contamination risks, and may reduce the risk of infectious disease transmission via breast milk.

The Microbiome of the Lower Respiratory Tract in Premature Infants with and without Severe Bronchopulmonary Dysplasia.

Aim We determined whether the bacteria in the lower respiratory tract (LRT) in extremely premature infants with severe bronchopulmonary dysplasia (BPD) are different from those with nonsevere BPD. Study Design We conducted a retrospective study of extremely premature infants who were admitted to the neonatal intensive care unit of Fukushima Medical University Hospital, Japan between April 2005 and March 2014. We screened for the bacterial colonization of the LRT using tracheobronchial aspirate fluid. Results A total of 169 extremely premature infants were included. Overall, 102 did not experience severe BPD, whereas the remaining 67 experienced severe BPD. Corynebacterium species (Cs) were more frequently detected in the severe BPD than nonsevere BPD infants (p = 0.03). There were significant differences between infants with and without severe BPD in the duration of endotracheal ventilation (p = 0.00, odds ratio [OR], 1.03; 95% confidence interval [CI], 1.01-1.06), the duration of supplemental oxygen (p = 0.00, OR, 1.02; 95% CI, 1.01-1.03) before 36 weeks of postmenstrual age, and the frequency of sepsis after 7 postnatal days (p = 0.01, OR, 1.73; 95% CI, 1.18-2.54). Conclusion Cs are more likely to be present in the severe BPD infants with longer duration of endotracheal ventilation.

Inhibition of cathepsin S reduces allogeneic T cell priming but not graft-versus-host disease against minor histocompatibility antigens.

Cathepsin (Cathepsin) S, L, and B proteases mediate antigen presentation on major histocompatibility complex (MHC) class II by degrading the invariant chain Ii, which blocks peptide loading. The ability of the Cathepsin S inhibitor LHVS (morpholinurea-leucine-homophenylalanine-vinylsulfone phenyl) to impede antigen presentation has led its development as a therapy for autoimmune diseases. There is substantial evidence that donor T cell recognition of host minor histocompatibility antigens (miHA) and subsequent destruction of host tissue mediates graft-versus-host disease (GVHD). We hypothesized that enzymes involved in antigen presentation may play a role in the development of GVHD. Using the C57BL/6 → BALB.B minor mismatch acute GVHD (aGVHD) model, we found that the cathepsin S activity of spleens from allogenetically transplanted mice were significantly increased 1 week after transplantation compared with syngeneic mice. Although LHVS decreased T cell priming responses against both single OVA antigen and miHA in vitro, LHVS did not reduce the severity of aGVHD. In fact, LHVS exacerbated a CD4(+)-T cell-dependent model of GVHD similar to chronic GVHD. This suggests that cytokines rather than T cells may mediate much of the damage in the aGVHD model and that therapeutics based on inhibition of antigen presentation for GVHD must be approached with caution.

Enzyme replacement therapy (ERT) procedure for mucopolysaccharidosis type II (MPS II) by intraventricular administration (IVA) in murine MPS II.

Mucopolysaccharidosis type II (MPS II), or Hunter syndrome, is a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS) and is characterized by the accumulation of glycosaminoglycans (GAGs). MPS II has been treated by hematopoietic stem cell therapy (HSCT)/enzyme replacement therapy (ERT), but its effectiveness in the central nervous system (CNS) is limited because of poor enzyme uptake across the blood-brain barrier (BBB). To increase the efficacy of ERT in the brain, we tested an intraventricular ERT procedure consisting of repeated administrations of IDS (20 µg/mouse/3 weeks) in IDS-knockout, MPS II model mice. The IDS enzyme activity and the accumulation of total GAGs were measured in mouse brains. The IDS activity was significantly increased, and the accumulation of total GAGs was decreased in the MPS II mouse brains treated with multiple administrations of IDS via intraventricular ERT. Additionally, a high level of IDS enzyme activity was appreciated in other MPS II mouse tissues, such as the liver, spleen, testis and others. A Y-maze was used to test learning and memory after repeated intraventricular ERT with IDS. The IDS-treated mouse groups recovered the capacity for short-term memory and activity. Although large and small vacuoles were found at the margin of the cerebellar Purkinje cells in the disease-control mice, these vacuoles disappeared upon treated with IDS. Loss of vacuoles was also observed in other tissues (liver, kidney and testis). These results demonstrate the possible efficacy of an ERT procedure with intraventricular administration of IDS for the treatment of MPS II.

Describing and Quantifying Wrong-Patient Medication Errors Through a Study of Incident Reports.

Purpose: Our aim was to inform a new definition of wrong-patient errors, obtained through an analysis of incident reports related to medication errors. Methods: We investigated wrong-patient medication errors in incident reports voluntarily reported by medical staff using a web-based incident reporting system from 2015 to 2016 at a university hospital in Japan. Incident report content was separately evaluated by four evaluators using investigational methods for clinical incidents from the Clinical Risk Unit and the Association of Litigation and Risk Management. They investigated whether it was the patient or drug that was incorrectly chosen during wrong-patient errors in drug administration in incident reports and assessed contributory factors which affected the error occurrence. The evaluators integrated the results and interpreted them together. Results: Out of a total 4337 IRs, only 30 cases (2%) contained wrong-patient errors in medication administration. The cases where the intended drugs were administered to incorrect patients occurred less frequently than cases where the wrong drugs were administered to the intended patients through the investigation of wrong targets. After a discussion, the evaluators concluded that the patient - drug/CPOE screen mismatch, caused by choosing the wrong patient, drug, or CPOE screen (mix-ups), occurred in the wrong-patient medication errors. These errors were caused by three conditions: (1) where two patients/drugs were listed next to one another, (2) where two patients' last names/drugs' names were the same, and (3) where the patient/drug/CPOE screen in front of the staff involved was believed to be the correct one. Additionally, these errors also involved insufficient confirmation, which led to failure to detect and correct the mismatch occurrences. Conclusion: Based on our study, we propose a new definition of wrong-patient medication errors: they consisted of choosing a wrong target and insufficient confirmation. We will investigate other types of wrong-patient errors to apply this definition.

Endoplasmic reticulum stress induces autophagy through activation of p38 MAPK in fibroblasts from Pompe disease patients carrying c.546G>T mutation.

Pompe disease (glycogen storage disease type II) is an autosomal recessive myopathic disorder arising from the deficiency of lysosomal acid α-glucosidase (GAA). Activation of autophagy is a key pathophysiological feature in skeletal muscle fibers and fibroblasts from patients with Pompe disease. The accumulation of autophagic vacuoles has been shown to interfere with the efficacy of enzyme replacement therapy with recombinant human GAA. However, the induction mechanism of autophagy in Pompe disease is still unclear. In this study, we show that misfolded GAA-induced endoplasmic reticulum (ER) stress triggers autophagy in a manner regulated by p38 MAPK signaling pathways in fibroblasts from late-onset patients with Pompe disease. By studying normal fibroblasts and patient fibroblasts carrying a c.546G>T mutation, we uncovered that mutant GAA was rapidly degraded by proteasome. In addition, we found both activation of ER stress response and autophagy in these patient fibroblasts. Treatment with N-butyl-deoxynojirimycin (NB-DNJ), which acts as a pharmacological chaperone for certain mutant forms of GAA, led to attenuation of not only ER stress, but also autophagy in patient fibroblasts. Levels of phosphorylated p38 MAPK observed in patient fibroblasts were decreased after treatment with NB-DNJ. The autophagic response in patient fibroblasts was also negatively regulated by treatment with the p38 MAPK inhibitor SB203580. These findings define a critical role for ER stress in the activation of autophagy due to GAA mutation, and provide evidence that chaperone therapy may be a useful treatment for alleviation of autophagy in Pompe disease patients carrying a chaperon-responsive mutation.

Generation of induced pluripotent stem (iPS) cells derived from a murine model of Pompe disease and differentiation of Pompe-iPS cells into skeletal muscle cells.

Our study is the first to demonstrate the ability to generate iPS cells from a mouse model of Pompe disease. Initially, mouse tail tip fibroblasts were harvested from male, 8-week-old (GAA) knockout mice, and three reprogramming factors (Oct3/4, Sox2 and Klf4) were transfected into the isolated donor cells using a retroviral vector. These iPS cells also showed decreased levels of GAA enzymatic activity and strong positive staining with periodic acid-Schiff (indicating the accumulation of glycogen) and acid phosphatase (lysosomal activation marker). Pompe-iPS cells were differentiated into skeletal muscle cells in Matrigel®-coated plates. Spindle-shaped skeletal muscle cells were successfully generated from Pompe-iPS cells and showed spontaneous contraction and positive staining with the myosin heavy chain antibody. Electron microscopic analysis of the skeletal muscle cells showed typical morphological features, including Z-bands, I-bands, A-bands and H-bands, which were visible in wild-type and Pompe cells. Furthermore, Pompe skeletal muscle cells accumulated massive glycogen in lysosomes. This study indicates that the iPS and skeletal muscle cells generated in this study could also be a useful disease model for studies investigating the pathogenesis and treatment of skeletal muscle in Pompe disease.

Pathogenesis of venous hypertrophy associated with schistosomiasis in whooper swans (Cygnus cygnus) in Japan.

Thirteen whooper swans (Cygnus cygnus) affected with schistosomiasis were examined pathologically. Venous hypertrophy, characterized by marked nodular proliferation of medial smooth muscle fibers with frequent obliteration of the vascular lumen, was observed in eight of the 13 whooper swans. Venous hypertrophy was located in the medium-sized veins of the mesentery, the serosa, and the muscular layer of the duodenum, jejunum, ileum, and cecum. In addition, vascular lesions were seen in the capsule and parenchymal interstitia of the liver, spleen, kidney, heart, aorta, air sac, and pleura. In mild lesions, segmental proliferation of medial smooth muscles was observed in the venous medium of the mesentery and serosa. Moderate lesions had a proliferation of smooth muscles in the veins with obliteration of venous lumens. In marked lesions, more severe proliferation of veins extended into the intestinal muscular layers and depressed them. Schistosome parasites were found in the venous lumens of each of the eight whooper swans with vascular lesions. Bile pigments and hemosiderin were observed in the livers of whooper swans. In addition, adult nematodes (Sarconema sp.) were localized in the myocardium of four of the eight whooper swans. The venous hypertrophy may be caused by the proliferation of medial smooth muscle fibers induced by schistosomiasis.

Polyhydramnios and abnormal foetal heart rate patterns in a foetus with Prader-Willi syndrome: A case report.

INTRODUCTION: Prader-Willi syndrome (PWS) is a complex neurodevelopmental genetic disorder. No definitive clinical signs of antenatal PWS have been identified. CASE: A healthy, nulliparous, 29-year-old woman demonstrated polyhydramnios at 27 weeks of gestation. Cardiotocography (CTG) showed an absence of foetal heart rate (FHR) acceleration and moderate FHR variability. Daily CTG demonstrated an absence of FHR acceleration. A male newborn was delivered by caesarean section, weighing 2492 g, which is appropriate for gestational age; the Apgar scores at 1 and 5 min were 6 and 6, respectively, and the umbilical artery pH was 7.295. The newborn exhibited marked hypotonia, lack of sucking, and cryptorchidism. FISH analysis performed due to severe hypotonia showed 46, XY. Ish del (15) (q11. 2q 11.2), which led to the diagnosis of PWS. DISCUSSION: Polyhydramnios and abnormal FHR patterns may be associated with feeding difficulty and hypotonia. These signs may be an indication for antenatal molecular genetic testing to diagnose PWS.

Draft Genome Sequence of Saccharomyces cerevisiae Strain P-684, Isolated from Prunus verecunda.

Saccharomyces cerevisiae strain P-684 is a yeast isolated from the flowers of Prunus verecunda 'Antiqua,' producing high quantities of malic and succinic acids in sake brewing. Here, we report the draft genome sequence of P-684, enlightening the mechanisms of biosynthesis of these organic acids by this strain.

Association Between Neutralizing Antibody Titers against Parechovirus A3 in Maternal and Cord Blood Pairs and Perinatal Factors.

BACKGROUND: Parechovirus A3 (PeV-A3) is a pathogen that causes severe infectious diseases such as sepsis and meningoencephalitis in neonates and young infants. In this study, we aimed to measure the neutralizing antibody titer (NAT) against PeV-A3 in paired maternal and cord blood samples and to clarify the serum epidemiology of PeV-A3 and the association between the NAT and perinatal factors. METHODS: NATs against PeV-A3 were measured in 1033 mothers (maternal and cord blood pairs; total of 2066 samples) who delivered their infant in Fukushima Prefecture between December 2013 and June 2014. RD-18S cells were used to measure NATs against PeV-A3. The association between NATs against PeV-A3 in maternal and cord blood and perinatal factors was determined using multivariate logistic regression analysis. RESULTS: The median gestational age of the infants was 39 weeks 4 days (interquartile range, 38 weeks 4 days to 40 weeks 3 days). The NATs against PeV-A3 in maternal blood and in cord blood were almost the same. The proportion of samples assigned to the low-titer group (NAT ≤ 1:16) was approximately 70%, and the proportion of samples assigned to the high-titer group tended to increase with gestational age. The high-titer rate and geometric mean titers decreased with increased maternal age. CONCLUSIONS: Cord blood indicates that neonates born at a lower gestational age and older mothers have a low NAT against PeV-A3. Thus, more attention should be paid to the onset of severe PeV-A3 disease in such neonates and young infants.

Bacterial and Fungal Counts of Dried and Semi-Dried Foods Collected from Dhaka, Bangladesh, and Their Reduction Methods.

　Food is a basic necessity for human survival, but it is still the vehicle for the transmission of food borne disease. Various studies have examined the roles of spices, herbs, nuts, and semi-dried fruits, making the need for safe and convenient methods of decontamination a necessity. The current study determined the bacterial and fungal loads of 26 spices and herbs, 5 nuts, 10 semi-dried fruits and 5 other foods. Spices, herbs and semi-dried foods demonstrated the highest bacterial and fungal loads with the majority showing over 104 CFU/mL. Nuts and other foods showed growths ranging from 102 to 106 CFU/mL. The current study also attempted to determine the effects of heat and plasma treatment. The log reduction of bacterial growth after heat treatment (maximum: 120 min for 60℃) was between 0.08 to 4.47, and the log reduction after plasma treatment (maximum: 40 min) ranged from 2.37 to 5.75. Spices showed the lowest rates of reduction, whereas the semi-dried and other foods showed moderate to high levels of decrease after heat treatment. The log reduction of fungal growth after heat treatment ranged from 0.27 to 4.40, and log reduction after plasma treatment ranged from 2.15 to 5.91．Furthermore, we validated the sterilization effect of plasma treatment against Bacillus spp. and Staphylococcus spp. by using scanning electron microscopy. Both treatment methods could prove to be advantageous in the agriculture related fields, enhancing the quality of the foods.

High plasma brain natriuretic polypeptide level as a marker of risk for thromboembolism in patients with nonvalvular atrial fibrillation.

BACKGROUND AND PURPOSE: Assessment of left atrial appendage (LAA) function with transesophageal echocardiography is useful for detecting patients at high risk for thromboembolism as a result of atrial fibrillation (AF). A recent study reported that the atrium is the main source of brain natriuretic polypeptide (BNP) in AF patients without overt heart failure. The purpose of this study was to assess a possible relationship between LAA function and plasma BNP levels in nonvalvular AF. METHODS: Thirty-four consecutive patients with chronic nonvalvular AF (age, 69+/-9 years) underwent transesophageal echocardiography and plasma BNP measurement. Thirteen patients with a history of thromboembolism or echocardiographic evidence of thrombus (E + group) were compared with 21 AF patients without complications (E- group). RESULTS: The E+ group patients demonstrated greater impairment of LAA velocity and higher plasma BNP levels than the E- group patients (LAA velocity: 12+/-6 versus 31+/-17 cm/s, P<0.05; plasma BNP: 126+/-53 versus 86+/-45 ng/L, P<0.05). Overall analysis of the continuous variables with multiple logistic regression analysis revealed that BNP was a significant predictor of thromboembolism. There was a weak but significant negative correlation between plasma BNP levels and LAA flow velocity (r=0.38, P<0.05). No intergroup difference in plasma atrial natriuretic polypeptide levels was found. CONCLUSIONS: The present data suggest the usefulness of measuring plasma BNP levels, which may reflect augmented atrial secretion of BNP from the impaired atrial myocardium, in detecting patients at high risk for thromboembolic complications in nonvalvular AF.

Pulmonary venous systolic flow: influence of gravity on pulmonary venous flow velocities assessed in patients with atrial fibrillation.

The origin of the pulmonary venous (PV) systolic flow wave is still unclear and could be the atrial relaxation and systolic descent of the atrioventricular plane, which decrease atrial pressure (suction) or raised PV pressure. In atrial fibrillation (AF), loss of atrial contraction and relaxation significantly modifies the systolic PV flow wave. The effect of recumbent positional changes on PV, however, has not yet been characterized in AF. The purpose of this study was to evaluate the effect of positional changes on systolic PV flow in patients with AF studied by transesophageal echocardiography. The study group consisted of 45 patients with AF (34 patients with AF, alone, and 11 patients with mitral stenosis [MS]). To assess the influence of left atrial pressure, we included patients with MS and AF. Pulsed wave Doppler transesophageal echocardiography of the left and right upper PV were performed in the left lateral recumbent position in all patients and repeated records were obtained with the subject in the supine position in 25 (AF alone: n = 20, MS: n = 5) of 45 patients. In the left lateral recumbent position, the systolic PV flow velocity and systolic fraction of the left PV, which were recorded on the recumbent subject's lower side, were significantly increased compared with those of the right PV in both AF alone and MS with AF (33.9 +/- 10.8 vs 13.8 +/- 6.4 cm/s, 0.45 +/- 0.09 vs 0.20 +/- 0.10 in AF alone; 30.2 +/- 11.7 vs 14.6 +/- 6.0 cm/s, 0.43 +/- 0.12 vs 0.20 +/- 0.07 in MS, respectively, P < .01). By changing the position from the left lateral to the supine position, systolic PV flow velocity and systolic fraction of the left and right PV became the same (29.3 +/- 8.4 vs 27.9 +/- 8.4 cm/s, 0.39 +/- 0.09 vs 0.36 +/- 0.06 in AF alone, 23.5 +/- 8.8 vs 27.5 +/- 5.0 cm/s, 0.35 +/- 0.08 vs 0.35 +/- 0.09 in MS, respectively). These findings show that the PV volume (hydrostatic pressure) significantly modifies systolic PV flow wave in patients without atrial contraction and relaxation. We should take into consideration the body position on which PV flow is studied.

New insights into the mechanism of the elevation of plasma brain natriuretic polypeptide levels in patients with left ventricular hypertrophy.

BACKGROUND: Messenger RNA of brain natriuretic polypeptide (BNP) is detected in both the atrium and the ventricle in vitro. Clinical usefulness has been shown in assessment of BNP level in patients with left ventricular hypertrophy; however, few studies have reported the role of the atrium and ventricle separately in the secretion of BNP from the hypertrophied heart. OBJECTIVE: To investigate how the atrium and ventricle secrete natriuretic peptides by comparing the regional concentration of atrial natriuretic polypeptide (ANP) or BNP in the hypertrophied heart with clinical parameters. PATIENTS AND METHODS: ANP and BNP were measured in blood samples from the aortic root, the anterior interventricular vein (AIV) and the coronary sinus in 12 control subjects, 10 subjects with hypertensive hypertrophy and eight with non-obstructive hypertrophic cardiomyopathy. The difference in concentration between the aortic root and the AIV and that between the AIV and the coronary sinus was calculated to estimate ventricular and atrial secretion, respectively. RESULTS: Plasma BNP levels correlated significantly with left ventricular mass index, pulmonary artery wedge pressure, stroke volume and left atrial dimension. Stepwise multiple regression analysis identified BNP from the atrium, not the ventricle, as an independent predictor of left ventricular mass. CONCLUSIONS: These data suggest that atrium-derived BNP is a significant predictor of left ventricular mass index in patients with left ventricular hypertrophy. The atrium-derived component contributes significantly to the elevation of plasma BNP level, reflecting atrial pressure and volume loading in left ventricular hypertrophy without systolic dysfunction.

Influence of Cardiac Pacing Mode on Left Atrial Appendage Flow Velocity: Implication to Systemic Embolism During VVI Pacing.

Previous studies have shown that the incidence of thromboembolism is higher in patients with single-chamber ventricular demand (VVI) pacemakers than in patients with dual-chamber (DDD) pacemakers. However, data on left atrial appendage flow velocity in pacing patients are limited. To investigate the influence of the pacing mode on the left atrial appendage flow velocity, we studied 19 patients with permanent DDD pacemakers and measured the left atrial appendage flow velocity by transesophageal echocardiography at baseline (during DDD pacing) and after switching to VVI pacing. The indications for pacemaker implantation were second- and third-degree atrioventricular block (AVB group, n = 11) and sick sinus syndrome (SSS group, n = 8). Compared with the DDD pacing mode, there was a significant decrease in the left atrial appendage flow velocity during VVI pacing in both the SSS group (43 +/- 14 vs 23 +/- 7 cm/sec, P < 0.05) and the AVB group (59 +/- 18 vs 41 +/- 18 cm/sec, P < 0.05). In eight patients with persistent retrograde ventriculoatrial conduction during VVI pacing, the left atrial appendage flow velocity was markedly decreased (from 43 +/- 16 to 25 +/- 9 cm/sec, P < 0.05). In five (63%) of the eight patients, left atrial appendage flow velocity was less than 25 cm/sec. A reduction in left atrial appendage flow velocity when switching from DDD to VVI pacing may account for an increased risk of thrombus formation in the left atrial appendage (an increased thromboembolic risk in patients in sinus rhythm with VVI pacemakers).

First isolation of border disease virus in Japan is from a pig farm with no ruminants.

The first isolation of border disease virus (BDV) in Japan was from a pig farm of the farrow-to-finishing type that kept no small ruminants or cattle. The infection was detected in the course of sero-surveillance for classical swine fever virus (CSFV) in Japan. The infected pigs had no clinical symptoms of CSFV or other disease; nevertheless, a high sero-positive rate of 58.5% was identified. A persistently infected pig with the BDV was found and suspected to be the cause of sero-prevalence in the farm. The isolated BDV was genetically close to BDV strains from New Zealand, but there was no epidemiological evidence concerning the route(s) of the invasion into the farm.

Biomarkers in newly diagnosed pediatric-extensive chronic graft-versus-host disease: a report from the Children's Oncology Group.

Numerous chronic graft-versus-host disease (cGVHD) biomarkers have been identified in limited, single-institution studies without validation. We hypothesized that plasma-derived biomarkers could diagnose, classify, and evaluate response in children with cGVHD. We performed a concomitant analysis of a number of known and predicted peripheral blood cGVHD biomarkers from a Children's Oncology Group (COG) phase 3 cGVHD therapeutic trial. A total of 52 newly diagnosed patients with extensive cGVHD were compared for time of onset after blood and marrow transplantation (BMT) (early, 3-8 months; late, > or = 9 months) with 28 time-matched controls with no cGVHD (early, 6 months after BMT; late, 12 months after BMT). Soluble B-cell activation factor (sBAFF), anti-dsDNA antibody, soluble IL-2 receptor alpha (sIL-2Ralpha), and soluble CD13 (sCD13) were elevated in patients with early-onset cGVHD compared with controls. sBAFF and anti-dsDNA were elevated in patients with late-onset cGVHD. Some of the biomarkers correlated with specific organ involvement and with therapeutic response. These 4 biomarkers had high specificity with higher sensitivity in combination. Changes in biomarker concentrations with immune reconstitution after transplantation significantly affected interpretation of results. The identified biomarkers have the potential for improved classification, early response evaluation, and direction of cGVHD treatment, but require validation in larger studies. This study is registered at www.cancer.gov/clinicaltrials as no. COG-ASCT0031.

Altered Toll-like receptor 9 responses in circulating B cells at the onset of extensive chronic graft-versus-host disease.

B cells appear to play a role in chronic graft-versus-host disease (cGVHD) as shown in murine models and the success of anti-CD20 B cell antibody treatment in humans. Recent studies have shown that immunostimulatory microbial CpG-DNA splenic responses were enhanced in murine GVHD. We hypothesized that CpG-induced B cell responses are increased in human cGVHD. Newly diagnosed cGVHD patients enrolled on the COG protocol ASCT0031 were divided into early (3-8 months postblood and marrow transplant [BMT]) and late (> or =9 months post-BMT) onset groups and compared to time-matched control BMT patients. A significantly greater percentage of phosphorothioate (PS)-modified CpG stimulated B cells from cGVHD patients demonstrated an increased expression of CD86 compared to controls (P = .0004). This response had a significant correlation between B cell TLR9 expression (r(2) = 0.65; P = .002) and CD86 upregulation using the entirely TLR9-dependent native phosphodiester CpG (P = .003). The PS-modified CpG response at 2 months after initiation of cGVHD therapy demonstrated a trend toward predicting therapeutic response at 9 months post-BMT (P = .07). These findings suggest that an increased number of B cells, primed for a TLR9 response, may play a role in the pathophysiology of cGVHD.