Ruxolitinib for Glucocorticoid-Refractory Acute Graft-versus-Host Disease.

BACKGROUND: Acute graft-versus-host disease (GVHD) remains a major limitation of allogeneic stem-cell transplantation; not all patients have a response to standard glucocorticoid treatment. In a phase 2 trial, ruxolitinib, a selective Janus kinase (JAK1 and JAK2) inhibitor, showed potential efficacy in patients with glucocorticoid-refractory acute GVHD. METHODS: We conducted a multicenter, randomized, open-label, phase 3 trial comparing the efficacy and safety of oral ruxolitinib (10 mg twice daily) with the investigator's choice of therapy from a list of nine commonly used options (control) in patients 12 years of age or older who had glucocorticoid-refractory acute GVHD after allogeneic stem-cell transplantation. The primary end point was overall response (complete response or partial response) at day 28. The key secondary end point was durable overall response at day 56. RESULTS: A total of 309 patients underwent randomization; 154 patients were assigned to the ruxolitinib group and 155 to the control group. Overall response at day 28 was higher in the ruxolitinib group than in the control group (62% [96 patients] vs. 39% [61]; odds ratio, 2.64; 95% confidence interval [CI], 1.65 to 4.22; P<0.001). Durable overall response at day 56 was higher in the ruxolitinib group than in the control group (40% [61 patients] vs. 22% [34]; odds ratio, 2.38; 95% CI, 1.43 to 3.94; P<0.001). The estimated cumulative incidence of loss of response at 6 months was 10% in the ruxolitinib group and 39% in the control group. The median failure-free survival was considerably longer with ruxolitinib than with control (5.0 months vs. 1.0 month; hazard ratio for relapse or progression of hematologic disease, non-relapse-related death, or addition of new systemic therapy for acute GVHD, 0.46; 95% CI, 0.35 to 0.60). The median overall survival was 11.1 months in the ruxolitinib group and 6.5 months in the control group (hazard ratio for death, 0.83; 95% CI, 0.60 to 1.15). The most common adverse events up to day 28 were thrombocytopenia (in 50 of 152 patients [33%] in the ruxolitinib group and 27 of 150 [18%] in the control group), anemia (in 46 [30%] and 42 [28%], respectively), and cytomegalovirus infection (in 39 [26%] and 31 [21%]). CONCLUSIONS: Ruxolitinib therapy led to significant improvements in efficacy outcomes, with a higher incidence of thrombocytopenia, the most frequent toxic effect, than that observed with control therapy. (Funded by Novartis; REACH2 ClinicalTrials.gov number, NCT02913261.).

Immunohistochemical markers for histopathological diagnosis and differentiation of acute cutaneous graft-versus-host disease.

Graft-versus-host disease (GvHD) is a major complication following stem-cell or solid-organ transplantation. Accurate diagnosis of cutaneous GvHD is challenging, given that drug eruptions and viral rashes may present with similar clinical/histological manifestations. Specific markers are not available. We performed the histological examination of biopsy samples from acute GvHD (aGvHD; n = 54), Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN; n = 27), maculopapular drug eruption (MDE; n = 26) and healthy controls (n = 26). Samples of aGvHD showed a decrease in Langerhans cells (LC, p = 0.0001) and an increase in macrophages (MΦ, p = 0.0001) compared to healthy skin. Compared to SJS/TEN, MDE and healthy skin, aGvHD biopsies contained greater numbers of CD4+ and CD8+ T cells. The majority of CD4+ T-helper cells were localized in the upper dermis, whereas cytotoxic CD8+ T cells were found in the epidermis. Increased numbers of CD56+ natural killer (NK) cells in the upper dermis of aGvHD skin (p = 0.007) were not observed in controls or SJS/TEN and MDE. There were no differences in elafin staining between aGvHD and the latter two conditions. Acute GvHD appears to have a distinct inflammatory cell profile (T cells/NK cells) that may aid establishing in a more accurate diagnosis, especially when used to rule out differential diagnoses such as SJS/TEN or MDE.

Increased age-associated mortality risk in HLA-mismatched hematopoietic stem cell transplantation.

We investigated a possible interaction between age-associated risk and HLA-mismatch associated risk on prognosis in different age categories of recipients of unrelated hematopoietic stem cell transplants (HSCT) (n=3019). Patients over 55 years of age transplanted with 8/10 donors showed a mortality risk of 2.27 (CI 1.70-3.03, P<0.001) and 3.48 (CI 2.49-4.86, P<0.001) when compared to 10/10 matched patients in the same age group and to 10/10 matched patients aged 18-35 years, respectively. Compared to 10/10 matched transplantations within each age category, the Hazards Ratio for 8/10 matched transplantation was 1.14, 1.40 and 2.27 in patients aged 18-35 years, 36-55 and above 55 years. Modeling age as continuous variable showed different levels of risk attributed to age at the time of transplantation [OS: 10/10: Hazards Ratio 1.015 (per life year); 9/10: Hazards Ratio: 1.019; 8/10: Hazards Ratio 1.026]. The interaction term was significant for 8/10 transplantations (P=0.009). Findings for disease-free survival and transplant-related mortality were similar. Statistical models were stratified for diagnosis and included clinically relevant predictors except cytomegalovirus status and Karnofsky performance status. The risk conferred by age at the time of transplantation varies according to the number of HLA-mismatches and leads to a disproportional increase in risk for elderly patients, particularly with double mismatched donors. Our findings highlight the importance of HLA-matching, especially in patients over 55 years of age, as HLA-mismatches are less well tolerated in these patients. The interaction between age-associated risk and HLA-mismatches should be considered in donor selection and in the risk assessment of elderly HSCT recipients.

Human epidermal Langerhans cells replenish skin xenografts and are depleted by alloreactive T cells in vivo.

Epidermal Langerhans cells (LC) are potent APCs surveying the skin. They are crucial regulators of T cell activation in the context of inflammatory skin disease and graft-versus-host disease (GVHD). In contrast to other dendritic cell subtypes, murine LC are able to reconstitute after local depletion without the need of peripheral blood-derived precursors. In this study, we introduce an experimental model of human skin grafted to NOD-SCID IL2Rγ(null) mice. In this model, we demonstrate that xenografting leads to the transient loss of LC from the human skin grafts. Despite the lack of a human hematopoietic system, human LC repopulated the xenografts 6 to 9 wk after transplantation. By staining of LC with the proliferation marker Ki67, we show that one third of the replenishing LC exhibit proliferative activity in vivo. We further used the skin xenograft as an in vivo model for human GVHD. HLA-disparate third-party T cells stimulated with skin donor-derived dendritic cells were injected intravenously into NOD-SCID IL2Rγ(null) mice that had been transplanted with human skin. The application of alloreactive T cells led to erythema and was associated with histological signs of GVHD limited to the transplanted human skin. The inflammation also led to the depletion of LC from the epidermis. In summary, we provide evidence that human LC are able to repopulate the skin independent of blood-derived precursor cells and that this at least partly relates to their proliferative capacity. Our data also propose xeno-transplantation of human skin as a model system for studying the role of skin dendritic cells in the efferent arm of GVHD.

Varicella-zoster virus glycoproteins B and E are major targets of CD4+ and CD8+ T cells reconstituting during zoster after allogeneic transplantation.

BACKGROUND: After allogeneic hematopoietic stem-cell transplantation patients are at increased risk for herpes zoster as long as varicella-zoster virus specific T-cell reconstitution is impaired. This study aimed to identify immunodominant varicella-zoster virus antigens that drive recovery of virus-specific T cells after transplantation. DESIGN AND METHODS: Antigens were purified from a varicella-zoster virus infected cell lysate by high-performance liquid chromatography and were identified by quantitative mass spectrometric analysis. To approximate in vivo immunogenicity for memory T cells, antigen preparations were consistently screened with ex vivo PBMC of varicella-zoster virus immune healthy individuals in sensitive interferon-γ ELISpot assays. Candidate virus antigens identified by the approach were genetically expressed in PBMC using electroporation of in vitro transcribed RNA encoding full-length proteins and were then analyzed for recognition by CD4(+) and CD8(+) memory T cells. RESULTS: Varicella-zoster virus encoded glycoproteins B and E, and immediate early protein 62 were identified in immunoreactive lysate material. Predominant CD4(+) T-cell reactivity to these proteins was observed in healthy virus carriers. Furthermore, longitudinal screening in allogeneic stem-cell transplantation patients showed strong expansions of memory T cells recognizing glycoproteins B and E after onset of herpes zoster, while immediate early protein 62 reactivity remained moderate. Reactivity to viral glycoproteins boosted by acute zoster was mediated by both CD4(+) and CD8(+) T cells. CONCLUSIONS: Our data demonstrate that glycoproteins B and E are major targets of varicella-zoster virus specific CD4(+) and CD8(+) T-cell reconstitution occurring during herpes zoster after allogeneic stem-cell transplantation. Varicella-zoster virus glycoproteins B and E might form the basis for novel non-hazardous zoster subunit vaccines suitable for immunocompromised transplant patients.

Presence of Microbial Contamination and Biofilms at a Beer Can Filling Production Line.

ABSTRACT: Contamination of beer arises in 50% of all events at the late stages of production, in the filling area. This is where biofilms, a consortia of microorganisms embedded in a matrix composed of extracellular polymeric substances, play a critical role. To date, most studies have focused on the presence of (biofilm-forming) microorganisms in the filling environment. Our aim was to characterize the microbial status as well as the presence of possible biofilms at a can filling line for beer by determining the presence of microorganisms and their associated matrix components (carbohydrates, proteins and extracellular DNA [eDNA]). For 23 sampling sites, targeted quantitative PCR confirmed the presence of microorganisms at 10 sites during operation and at 3 sites after cleaning. The evaluation of carbohydrates, eDNA, and proteins showed that 16 sites were positive for at least one component during operation and 4 after cleaning. We identified one potential biofilm hotspot, namely the struts below the filler, harboring high loads of bacteria and yeast, eDNA, carbohydrates, and proteins. The protein pattern was different from that of beer. This work deepens our understanding of biofilms and microorganisms found at the filling line of beer beverages at sites critical for production.

Bacteria of eleven different species isolated from biofilms in a meat processing environment have diverse biofilm forming abilities.

Biofilms are formed by microorganisms protected by a self-produced matrix, most often attached to a surface. In the food processing environments biofilms endanger the product safety by the transmission of spoilage and pathogenic bacteria. In this study, we characterised the biofilm formation of the following eleven strains isolated from biofilms in a meat-processing environment: Acinetobacter harbinensis BF1, Arthrobacter sp. BF1, Brochothrix thermosphacta BF1, Carnobacterium maltaromaticum BF1, Kocuria salsicia BF1, Lactococcus piscium BF1, Microbacterium sp. BF1, Pseudomonas fragi BF1, Psychrobacter sp. BF1, Rhodococcus erythropolis BF1, Stenotrophomonas sp. BF1. We applied whole- genome sequencing and subsequent genome analysis to elucidate genetic features associated with the biofilm lifestyle. We furthermore determined the motility and studied biofilm formation on stainless steel using a static mono-species biofilm model mimicking the meat processing environment. The biomass and the EPS components carbohydrates, proteins and extracellular DNA (eDNA) of the biofilms were investigated after seven days at 10 °C. Whole-genome analysis of the isolates revealed that all strains except the Kocuria salsicia BF1 isolate, harboured biofilm associated genes, including genes for matrix production and motility. Genes involved in cellulose metabolism (present in 82% of the eleven strains) and twitching motility (present in 45%) were most frequently found. The capacity for twitching was confirmed using plate assays for all strains except Lactococcus piscium BF1, which showed the lowest motility behaviour. Differences in biofilm forming abilities could be demonstrated. The bacterial load ranged from 5.4 log CFU/cm2 (Psychrobacter sp. isolate) to 8.7 log CFU/cm2 (Microbacterium sp. isolate). The amount of the matrix components varied between isolates. In the biofilm of six strains we detected all three matrix components at different levels (carbohydrates, proteins and eDNA), in two only carbohydrates and eDNA, and in three only carbohydrates. Carbohydrates were detected in biofilms of all strains ranging from 0.5 to 4.3 µg glucose equivalents/cm2. Overall, the Microbacterium sp. strain showed the highest biofilm forming ability with high bacterial load (8.7 log CFU/cm2) and high amounts of carbohydrates (2.2 µg glucose equivalents/cm2), proteins (present in all experiments) and eDNA (549 ng/cm2). In contrast, Brochothrix thermosphacta was a weak biofilm former, showing low bacterial load and low levels of carbohydrates in the matrix (6.2 log CFU/cm2 and 0.5 µg glucose equivalents/cm2). This study contributes to our understanding of the biofilm forming ability of bacteria highly abundant in the meat processing environment, which is crucial to develop strategies to prevent and reduce biofilm formation in the food producing environment.

Identification of biofilm hotspots in a meat processing environment: Detection of spoilage bacteria in multi-species biofilms.

Biofilms are comprised of microorganisms embedded in a self-produced matrix that normally adhere to a surface. In the food processing environment they are suggested to be a source of contamination leading to food spoilage or the transmission of food-borne pathogens. To date, research has mainly focused on the presence of (biofilm-forming) bacteria within food processing environments, without measuring the associated biofilm matrix components. Here, we assessed the presence of biofilms within a meat processing environment, processing pork, poultry and beef, by the detection of microorganisms and at least two biofilm matrix components. Sampling included 47 food contact surfaces and 61 non-food contact surfaces from eleven rooms within an Austrian meat processing plant, either during operation or after cleaning and disinfection. The 108 samples were analysed for the presence of microorganisms by cultivation and targeted quantitative real-time PCR based on 16S rRNA. Furthermore, the presence of the major matrix components carbohydrates, extracellular DNA and proteins was evaluated. Overall, we identified ten biofilm hotspots, among them seven of which were sampled during operation and three after cleaning and disinfection. Five biofilms were detected on food contact surfaces (cutters and associated equipment and a screw conveyor) and five on non-food contact surfaces (drains and water hoses) resulting in 9.3 % of the sites being classified as biofilm positive. From these biofilm positive samples, we cultivated bacteria of 29 different genera. The most prevalent bacteria belonged to the genera Brochothrix (present in 80 % of biofilms), Pseudomonas and Psychrobacter (isolated from 70 % biofilms). From each biofilm we isolated bacteria from four to twelve different genera, indicating the presence of multi-species biofilms. This work ultimately determined the presence of multi-species biofilms within the meat processing environment, thereby identifying various sources of potential contamination. Especially the identification of biofilms in water hoses and associated parts highlights the need of a frequent monitoring at these sites. The knowledge gained about the presence and composition of biofilms (i.e. chemical and microbiological) will help to prevent and reduce biofilm formation within food processing environments.

Role of Donor Clonal Hematopoiesis in Allogeneic Hematopoietic Stem-Cell Transplantation.

PURPOSE: Clonal hematopoiesis of indeterminate potential (CHIP) occurs in the blood of approximately 20% of older persons. CHIP is linked to an increased risk of hematologic malignancies and of all-cause mortality; thus, the eligibility of stem-cell donors with CHIP is questionable. We comprehensively investigated how donor CHIP affects outcome of allogeneic hematopoietic stem-cell transplantation (HSCT). METHODS: We collected blood samples from 500 healthy, related HSCT donors (age ≥ 55 years) at the time of stem-cell donation for targeted sequencing with a 66-gene panel. The effect of donor CHIP was assessed on recipient outcomes, including graft-versus-host disease (GVHD), cumulative incidence of relapse/progression (CIR/P), and overall survival (OS). RESULTS: A total of 92 clonal mutations with a median variant allele frequency of 5.9% were identified in 80 (16.0%) of 500 donors. CHIP prevalence was higher in donors related to patients with myeloid compared with lymphoid malignancies (19.2% v 6.3%; P ≤ .001). In recipients allografted with donor CHIP, we found a high cumulative incidence of chronic GVHD (cGVHD; hazard ratio [HR], 1.73; 95% CI, 1.21 to 2.49; P = .003) and lower CIR/P (univariate: HR, 0.62; 95% CI, 0.40 to 0.97; P = .027; multivariate: HR, 0.63; 95% CI, 0.41 to 0.98; P = .042) but no effect on nonrelapse mortality. Serial quantification of 25 mutations showed engraftment of 24 of 25 clones and disproportionate expansion in half of them. Donor-cell leukemia was observed in two recipients. OS was not affected by donor CHIP status (HR, 0.88; 95% CI, 0.65 to 1.321; P = .434). CONCLUSION: Allogeneic HSCT from donors with CHIP seems safe and results in similar survival in the setting of older, related donors. Future studies in younger and unrelated donors are warranted to extend these results. Confirmatory studies and mechanistic experiments are warranted to challenge the hypothesis that donor CHIP might foster cGVHD development and reduce relapse/progression risk.

Comparative genomics of human and non-human Listeria monocytogenes sequence type 121 strains.

The food-borne pathogen Listeria (L.) monocytogenes is able to survive for months and even years in food production environments. Strains belonging to sequence type (ST)121 are particularly found to be abundant and to persist in food and food production environments. To elucidate genetic determinants characteristic for L. monocytogenes ST121, we sequenced the genomes of 14 ST121 strains and compared them with currently available L. monocytogenes ST121 genomes. In total, we analyzed 70 ST121 genomes deriving from 16 different countries, different years of isolation, and different origins-including food, animal and human ST121 isolates. All ST121 genomes show a high degree of conservation sharing at least 99.7% average nucleotide identity. The main differences between the strains were found in prophage content and prophage conservation. We also detected distinct highly conserved subtypes of prophages inserted at the same genomic locus. While some of the prophages showed more than 99.9% similarity between strains from different sources and years, other prophages showed a higher level of diversity. 81.4% of the strains harbored virtually identical plasmids. 97.1% of the ST121 strains contain a truncated internalin A (inlA) gene. Only one of the seven human ST121 isolates encodes a full-length inlA gene, illustrating the need of better understanding their survival and virulence mechanisms.

Signaling abnormalities, apoptosis, and reduced proliferation of circulating and tumor-infiltrating lymphocytes in patients with oral carcinoma.

We have reported earlier that T cells found in the tumor microenvironment of head and neck cancer showed evidence of apoptosis as well as decreased expression of signaling molecules. In this prospective study, spontaneous apoptosis in tumor-infiltrating lymphocytes (TILs) and in paired circulating peripheral blood lymphocytes (PBLs) was evaluated in 28 patients with oral carcinoma and correlated with zeta-chain expression and anti-CD3 antibody-induced proliferation of the PBL obtained from each patient. In addition, expression of CD3, CD4, and CD8 molecules on TIL and Fas ligand (FasL) on the tumor was studied by immunohistochemistry. Soluble FasL was measured in the patients' sera. PBL obtained from 20 age-matched normal donors was used as a control. Reduced zeta-chain expression was observed in TIL-T of 9 of 28 patients and in PBL-T of 12 of 28 patients. Low zeta expression in autologous TIL-T and PBL-T was correlated (P < 0.0012), and it was associated with high levels of expression of FasL on the tumor (P = 0.0002 and P < 0.0013, respectively). Low zeta expression in PBL-T was also associated with the poor ability of these cells to proliferate in response to anti-CD3 antibodies (P = 0.0012). Increased proportions of apoptotic cells were detected in PBL of 6 of 28 (21%) patients versus 13 of 28 patients (46%) in TIL. Apoptosis in autologous PBL and TIL was found to correlate (P = 0.0322) and was significantly associated with reduced zeta-chain expression. Serum levels of soluble FasL were decreased in patients relative to normal controls but did not correlate with PBL apoptosis or FasL expression on the tumor. Decreased expression of TcR-associated zeta chain, depressed immune function, and apoptosis of T cells were observed to occur concomitantly in TIL and circulating PBL-T of a subset of patients with oral carcinoma. These alterations correlated with high levels of FasL expression on the tumor but not with the disease stage. The results suggest that tumor exerts systemic suppressive effects on immune cells, which may be, in part, mediated via the Fas/FasL pathway.

Superior in vitro stimulation of human CD8+ T-cells by whole virus versus split virus influenza vaccines.

Pandemic and seasonal influenza viruses cause considerable morbidity and mortality in the general human population. Protection from severe disease may result from vaccines that activate antigen-presenting DC for effective stimulation of influenza-specific memory T cells. Special attention is paid to vaccine-induced CD8+ T-cell responses, because they are mainly directed against conserved internal influenza proteins thereby presumably mediating cross-protection against circulating seasonal as well as emerging pandemic virus strains. Our study showed that influenza whole virus vaccines of major seasonal A and B strains activated DC more efficiently than those of pandemic swine-origin H1N1 and pandemic-like avian H5N1 strains. In contrast, influenza split virus vaccines had a low ability to activate DC, regardless which strain was investigated. We also observed that whole virus vaccines stimulated virus-specific CD8+ memory T cells much stronger compared to split virus counterparts, whereas both vaccine formats activated CD4+ Th cell responses similarly. Moreover, our data showed that whole virus vaccine material is delivered into the cytosolic pathway of DC for effective activation of virus-specific CD8+ T cells. We conclude that vaccines against seasonal and pandemic (-like) influenza strains that aim to stimulate cross-reacting CD8+ T cells should include whole virus rather than split virus formulations.

Safety and immunogenicity of a vero cell culture-derived whole-virus H5N1 influenza vaccine in chronically ill and immunocompromised patients.

The development of vaccines against H5N1 influenza A viruses is a cornerstone of pandemic preparedness. Clinical trials of H5N1 vaccines have been undertaken in healthy subjects, but studies in risk groups have been lacking. In this study, the immunogenicity and safety of a nonadjuvanted cell culture-derived whole-virus H5N1 vaccine were assessed in chronically ill and immunocompromised adults. Subjects received two priming immunizations with a clade 1 A/Vietnam H5N1 influenza vaccine, and a subset also received a booster immunization with a clade 2.1 A/Indonesia H5N1 vaccine 12 to 24 months later. The antibody responses in the two populations were assessed by virus neutralization and single radial hemolysis assays. The T-cell responses in a subset of immunocompromised patients were assessed by enzyme-linked immunosorbent spot assay (ELISPOT). The priming and the booster vaccinations were safe and well tolerated in the two risk populations, and adverse reactions were predominantly mild and transient. The priming immunizations induced neutralizing antibody titers of ≥1:20 against the A/Vietnam strain in 64.2% of the chronically ill and 41.5% of the immunocompromised subjects. After the booster vaccination, neutralizing antibody titers of ≥1:20 against the A/Vietnam and A/Indonesia strains were achieved in 77.5% and 70.8%, respectively, of chronically ill subjects and in 71.6% and 67.5%, respectively, of immunocompromised subjects. The T-cell responses against the two H5N1 strains increased significantly over the baseline values. Substantial heterosubtypic T-cell responses were elicited against the 2009 pandemic H1N1 virus and seasonal A(H1N1), A(H3N2), and B subtypes. There was a significant correlation between T-cell responses and neutralizing antibody titers. These data indicate that nonadjuvanted whole-virus cell culture-derived H5N1 influenza vaccines are suitable for immunizing chronically ill and immunocompromised populations. (This study is registered at ClinicalTrials.gov under registration no. NCT00711295.).