The oral microbiome of patients undergoing treatment for severe aplastic anemia: a pilot study.

The microbiome, an intriguing component of the human body, composed of trillions of microorganisms, has prompted scientific exploration to identify and understand its function and role in health and disease. As associations between microbiome composition, disease, and symptoms accumulate, the future of medicine hinges upon a comprehensive knowledge of these microorganisms for patient care. The oral microbiome may provide valuable and efficient insight for predicting future changes in disease status, infection, or treatment course. The main aim of this pilot study was to characterize the oral microbiome in patients with severe aplastic anemia (SAA) during their therapeutic course. SAA is a hematologic disease characterized by bone marrow failure which if untreated is fatal. Treatment includes either hematopoietic stem cell transplantation (HSCT) or immunosuppressive therapy (IST). In this study, we examined the oral microbiome composition of 24 patients admitted to the National Institutes of Health (NIH) Clinical Center for experimental SAA treatment. Tongue brushings were collected to assess the effects of treatment on the oral microbiome. Twenty patients received standard IST (equine antithymocyte globulin and cyclosporine) plus eltrombopag. Four patients underwent HSCT. Oral specimens were obtained at three time points during treatment and clinical follow-up. Using a novel approach to 16S rRNA gene sequence analysis encompassing seven hypervariable regions, results demonstrated a predictable decrease in microbial diversity over time among the transplant patients. Linear discriminant analysis or LefSe reported a total of 14 statistically significant taxa (p < 0.05) across time points in the HSCT patients. One-way plots of relative abundance for two bacterial species (Haemophilus parainfluenzae and Rothia mucilaginosa) in the HSCT group, show the differences in abundance between time points. Only one bacterial species (Prevotella histicola) was noted in the IST group with a p value of 0.065. The patients receiving immunosuppressive therapy did not exhibit a clear change in diversity over time; however, patient-specific changes were noted. In addition, we compared our findings to tongue dorsum samples from healthy participants in the Human Microbiome Project (HMP) database and found among HSCT patients, approximately 35% of bacterial identifiers (N = 229) were unique to this study population and were not present in tongue dorsum specimens obtained from the HMP. Among IST-treated patients, 45% (N = 351) were unique to these patients and not identified by the HMP. Although antibiotic use may have likely influenced bacterial composition and diversity, some literature suggests a decreased impact of antimicrobials on the oral microbiome as compared to their effect on the gut microbiome. Future studies with larger sample sizes that focus on the oral microbiome and the effects of antibiotics in an immunosuppressed patient population may help establish these potential associations.

Optimization of intrabone delivery of hematopoietic progenitor cells in a swine model using cell radiolabeling with [89]zirconium.

Intrabone (IB) hematopoietic cell transplantation (HCT) of umbilical cord blood in humans remains experimental and the technique has not been optimized. It is unknown whether hematopoietic progenitor cells (HPCs) injected IB are initially retained in the marrow or rapidly enter into the venous circulation before homing to the marrow. To develop an IB-injection technique that maximizes HPC marrow-retention, we tracked radiolabeled human HPCs following IB-injection into swine. We developed a method to radionuclide-label HPCs using a long-lived positron emitter (89) Zr and protamine sulfate that resulted in cellular-retention of low-dose radioactivity. This approach achieved radioactivity levels sufficient for detection by positron emission tomography with both high sensitivity and spatial resolution when fused with computed tomography. We found that conditions utilized in pilot IB-HCT clinical trials conducted by others led to both rapid drainage into the central venous circulation and cellular extravasation into surrounding muscle and soft tissues. By optimizing the needle design, using continuous real-time intra-marrow pressure monitoring, and by reducing the infusion-volume and infusion-rate, we overcame this limitation and achieved high retention of HPCs in the marrow. This method of IB cellular delivery is readily applicable in the clinic and could be utilized in future investigational IB-HCT trials aimed at maximizing marrow retention of HPCs.

Human papillomavirus reactivation following treatment of genital graft-versus-host disease.

Vaginal chronic graft-versus-host disease (cGVHD) is a common complication of stem cell transplantation. Human papillomavirus (HPV) disease can reactivate after transplantation, presumably because of immune factors affecting systemic immunity, such as waning antibody titers, impaired T- and B-lymphocyte responses, and the use of immunosuppressive therapies. However, a relationship between the use of local immunosuppressive agents and HPV reactivation and spread has not been previously described, to our knowledge. A 30-year-old woman, 2 years post transplant receiving systemic cyclosporine for cGVHD, was treated with vaginal dilators, topical corticosteroids, and estrogen for vaginal cGVHD. Colposcopy and biopsy for abnormal cytology revealed condylomatous cervicitis. Over the next 4 months, while continuing dilator therapy, linear verrucous lesions developed in the vagina and vulva, and were successfully treated with laser therapy. Use of local immunosuppression and dilators for genital GVHD can enhance spread of HPV infection. Integration of HPV screening and treatment into the care of women with genital cGVHD and development of strategies to manage both conditions simultaneously are warranted.

Three types of blood group I specificity among monoclonal anti-I autoantibodies revealed by analogues of a branched erythrocyte glycolipid.

Blood group I activities of the purified glycosphingolipid lacto-N-iso-octaosyl ceramide (Fromula: see text) and 8 of its analogues have been evaluated with 11 anti-I sera including 5 anti-I sera previously tested. All but one of the antisera were inhibited by the lacto-N-iso-octaosyl structure. Three types of I-specificity could be distinguished although none of the anti-I sera was identical in its inhibition patterns with the nine glycophingolipid analogues. The anti-I sera Ma and Woj represent the first type and require an intact Galbeta1 leads to 4GlcNAcbeta1 leads to 6 chain, the anti-I sera Step, Gra, Ver, and Ful represent the second type which requires Galbeta1 leads to 4GlcNAcbeta1 leads to 3 chain with branching, and the anti-I sera Phi, Da, Sch, and Low belong to the third type which requires both branches to be intact. Anti-I antibodies varry in their ability to react with their antigenic determinants in the presence of external substitutions with alpha-linked galactose or sialic acid.

Radioactive labeling of antibody: a simple and efficient method.

A simple and efficient method of covalently coupling the strong chelator diethylenetriaminepentaacetic acid to proteins was developed for radiolabeling immunoglobulin G antibodies. After being coupled and labeled with indium-111, a monoclonal antibody to carcinoembryonic antigen retained its ability to bind to its antigen in vitro and in vivo. In nude mice with a human colorectal xenograft, 41 percent of the injected radioactivity became localized in each gram of xenograft at 24 hours compared with 9 percent for control antibody and 19 percent for radioiodinated antibody to carcinoembryonic antigen.

Natural killer cell immunotherapy for cancer: a new hope.

Recently there has been a substantial gain in our understanding of the role NK-cells play in mediating innate host immune responses. Although NK cells have long been known to mediate antigen independent tumor cytotoxicity, the therapeutic potential of NK cell-based immunotherapy has yet to be realized. Manipulating the balance between inhibitory and activating NK receptor signals, sensitization of tumor target cells to NK cell-mediated apoptosis, and recent discoveries in NK-cell receptor biology have fueled translational research that has led to clinical trials investigating a number of novel methods to potentiate NK cytotoxicity against human malignancies.

Rapid natural killer cell recovery determines outcome after T-cell-depleted HLA-identical stem cell transplantation in patients with myeloid leukemias but not with acute lymphoblastic leukemia.

Natural killer (NK) cells are the first lymphocytes to recover after allogeneic stem cell transplantation (SCT) and can exert powerful graft-versus-leukemia (GVL) effects determining transplant outcome. Conditions governing NK cell alloreactivity and the role of NK recovery in sibling SCT are not well defined. NK cells on day 30 post-transplant (NK30) were measured in 54 SCT recipients with leukemia and donor and recipient killer immunoglobulin-like receptor (KIR) genotype determined. In univariate analysis, donor KIR genes 2DL5A, 2DS1, 3DS1 (positive in 46%) and higher numbers of inhibitory donor KIR correlated with higher NK30 counts and were associated with improved transplant outcome. NK30 counts also correlated directly with the transplant CD34 cell dose and inversely with the CD3+ cell dose. In multivariate analysis, the NK30 emerged as the single independent determinant of transplant outcome. Patients with NK30 >150/microl had less relapse (HR 18.3, P=0.039), acute graft-versus-host disease (HR 3.2, P=0.03), non-relapse mortality (HR 10.7, P=0.028) and improved survival (HR 11.4, P=0.03). Results suggest that T cell-depleted SCT might be improved and the GVL effect enhanced by selecting donors with favorable KIR genotype, and by optimizing CD34 and CD3 doses.

The second international meeting on allogeneic transplantation in solid tumors.

In October 2005, the second international meeting on allogeneic transplantation in solid tumors was convened in Stresa (Italy). The aim of this second meeting was to share clinical experiences of allografting in solid tumors, to discuss preclinical data on the mechanisms of graft-versus-tumor (GVT) effect, and to review methods for more efficacious transplant approaches. On the first day, the most recent results in cancer immunotherapy were reviewed; head-to head comparisons of clinical results achieved by standard therapy and by allografting in renal, breast, and ovarian cancer were presented. On the second day, GVT mechanisms and preclinical models were examined; anecdotal reports of a GVT effect in sarcoma, pancreatic cancer, prostate cancer, colorectal cancer and lung cancer were presented; new strategies for optimizing transplant outcome were discussed, including patient selection, tumor debulking, auto-allo approaches, selective T-cell depletion, targeting with monoclonal antibodies, use of killer cell immunoglobulin-like receptor-ligand mismatched natural killer cells. In conclusion, allografting in solid tumors is feasible with limited toxicities and transplant-related mortality; a GVT effect has been documented in many different solid tumors; targeting of the immune response to the tumor by new strategies and identification of the target antigen(s) of the GVT effect are promising areas of research.

Health-related quality of life in patients receiving reduced-intensity conditioning allogeneic hematopoietic stem cell transplantation.

Reduced-intensity conditioning allogeneic HSCT (RIC) has less regimen-related morbidity and mortality than myeloablative allogeneic HSCT (MT) offering allogeneic transplantation to patients otherwise excluded. Whether these advantages improve health-related quality of life (HRQL) is unknown. We examined the HRQL effects of RIC and MT in patients with hematological diseases pre-transplant (baseline), days 0, 30, 100, 1 and 2 years following HSCT. HRQL was measured using the Short Form-36 Health Survey and the Functional Assessment of Cancer Therapy - General and BMT. Data were analyzed using mixed linear modeling adjusting for baseline HRQL differences. Patients (RIC=41, MT=35) were predominately male (67%), in remission/stable disease (65%) with an Eastern Cooperative Oncology Group status

A pilot study of nonmyeloablative allogeneic hematopoietic stem cell transplant for advanced systemic mastocytosis.

Systemic mastocytosis (SM) is a disease characterized by tissue infiltration of neoplastic mast cells originating from hematopoietic stem cells. Patients with advanced SM have a poor prognosis, and there is no mast cell ablative therapy available for most patients who carry an activating point mutation in the c-kit gene. We report results of a prospective study evaluating the safety, engraftment, and possibility of inducing a graft-versus-mast cell (GvMC) effect after allogeneic nonmyeloablative hematopoietic cell transplantation (HCT) from an HLA-identical sibling. Three patients with advanced SM were transplanted. All achieved complete donor T cell chimerism followed by clinical evidence for GvMC effect. However, all patients experienced disease progression with the longest response duration of 39 months. The GvMC effect can be observed after nonmyeloablative HCT with limited efficacy. Effective cytoreductive therapy prior to HCT may be required for long-term disease control and cure.

Successful treatment of metastatic renal cell carcinoma with a nonmyeloablative allogeneic peripheral-blood progenitor-cell transplant: evidence for a graft-versus-tumor effect.

PURPOSE: A 50-year-old man developed progressive pulmonary metastasis resistant to interferon alfa-2b treatment 7 months after he underwent left nephrectomy for stage III renal cell carcinoma. We performed a nonmyeloablative allogeneic peripheral-blood stem-cell transplant in this patient to exploit a possible graft-versus-tumor effect from allogeneic lymphocytes. MATERIALS AND METHODS: The conditioning regimen consisted of fludarabine and cyclophosphamide followed by a T-cell replete, granulocyte-colony stimulating-factor-mobilized peripheral-blood stem-cell transplant from his HLA-identical brother. Cyclosporine was administered from days -4 to +45 to prevent graft rejection and acute graft-versus-host disease (GVHD). RESULTS: Serial polymerase chain reaction analysis of hematopoietic lineage-specific minisatellites initiallyshowed mixed chimerism in CD14(+) and CD15(+) myeloid cells, CD3(+) T cells, and CD34(+) progenitor cells, with rapid conversion to 100% donor T-cell chimerism by day +60 and 100% donor myeloid cells by day +100. Serial computed tomography scans of the chest showed stable disease at day +30, slight regression of pulmonary lesions at day +63, and complete disappearance of all pulmonary metastatic disease by day +110. Mild transient acute GVHD disease of the skin occurred on day +60 and limited chronic GVHD of the skin occurred by day +200. CONCLUSION: The complete regression of metastatic disease, which has now been maintained for more than 1 year, is compatible with a graft-versus-tumor effect.

Imatinib synergizes with donor lymphocyte infusions to achieve rapid molecular remission of CML relapsing after allogeneic stem cell transplantation.

Donor lymphocyte infusions (DLI) have been the mainstay of treatment for chronic myeloid leukemia (CML) relapsing after allogeneic stem cell transplantation (allo-SCT). Imatinib mesylate (IM) is also effective in these patients. However, advanced phase relapse (APRel) responds poorly with either treatment. To test the possibility that combinations of DLI and IM might be more effective, 37 patients with CML relapsing after allo-SCT between August 1994 and May 2004 were studied. Ten had molecular relapse (MRel), 14 hematological relapse (HRel) and 13 APRel. Thirteen received DLI, 9 IM and 11 DLI+IM. Four patients received DLI+IM but not concurrently. Thirty (81%) patients responded (actuarial survival and current leukemia-free survival of 80.6 +/- 6.7% and 69.1 +/- 7.7%). Of 30 patients, 26 are in molecular remission (MR), median follow-up of 1,226 days (range 249-3257) since relapse. Ten of 11 patients (including four with APRel) treated with DLI+IM achieved MR in 3 months and all are alive in MR. In contrast, only two of 22 treated with either modality (1/13 DLI and 1/9 IM) achieved MR at 3 months, 15 are alive, 11 in MR. Four patients receiving nonconcurrent DLI+IM are also alive in MR. In conclusion, DLI appears to synergize with IM to induce rapid and durable MR.

Nonmyeloablative blood stem cell transplantation as adoptive allogeneic immunotherapy for metastatic renal cell carcinoma.

Allogeneic stem cell transplantation has emerged as a potentially curative form of immunotherapy for patients with hematological malignancies that are resistant to conventional chemo/radiotherapy. Donor T cell populations targeting allogeneic minor histocompatibility antigens expressed on the patient's malignant cells are felt to be the driving force of the graft-versus-leukemia reaction, although to date only a handful of these antigens have been fully characterized. Recent data from experimental animal models and limited clinical data in humans suggest that graft-versus-tumor effects, analogous to the graft-versus-leukemia reaction, may be generated against malignancies of epithelial origin. This article reviews the results of a pilot trial demonstrating graft-versus-renal cell carcinoma effects following nonmyeloablative stem cell transplantation, highlighting the potential of allogeneic immunotherapy for treating cancer.

Monoclonal antibody against a cross-reactive idiotypic determinant found on human autoantibodies with anti-I and -i specificities.

A monoclonal antibody has been raised against a cross-reactive idiotypic determinant expressed on human autoantibodies with anti-I and -i specificities. The McAb is directed against a conformational epitope requiring interaction of H- and L-chains for maximal expression. This epitope was strongly expressed on the monoclonal protein of one out of 100 patients with paraproteinaemia and the peripheral blood lymphocytes of one out of 50 cases of B-cell leukaemia. A small amount of the epitope is detectable among immunoglobulins in normal serum.

Plasma vitamin D metabolites and C-reactive protein in stage-stop racing endurance sled dogs.

BACKGROUND: Dogs are a unique model for examining the effects of exercise on vitamin D status because of their lack of vitamin D synthesis by UV exposure. In addition, the inflammatory response may be associated with hypovitaminosis D. OBJECTIVES: To investigate the effects of several days of endurance exercise on plasma vitamin D (25-(OH)D3, 24,25-(OH)D3 and 1,25(OH)D3) and serum C-reactive protein (CRP) concentrations in stage-stop racing sled dogs. ANIMALS: 12 racing sled dogs and 8 control dogs. METHODS: Blood was collected before the race and immediately after racing on days 2 and 8. Plasma vitamin D metabolites and serum CRP concentrations were measured. RESULTS: Racing dogs showed a significant increase in 25(OH)D3 on day 2 (P = .027) and day 8 of the race (P < .001), whereas no increases were observed in control dogs. The plasma concentration of 24,25(OH)D3 showed a significant increase by day 8 (P < .001). There were no significant changes in 1,25(OH) D3 concentrations across all time points and groups. Racing dogs had significantly increased CRP concentrations by day 2 (39.3 ± 30.1 µg/mL; P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Increases in vitamin D metabolites as well as increases in CRP concentrations were observed in racing sled dogs. This finding was contrary to the hypothesis that decreases in vitamin D status in athletes may be related to the acute phase inflammatory response during exercise. In addition, the increased 24,25(OH)D3 concentrations compared to what is observed in other species suggests metabolic variations in dogs that lead to enhanced disposal of vitamin D.

Conditioning with rabbit versus horse ATG dramatically alters clinical outcomes in identical twins with severe aplastic anemia transplanted with the same allogeneic donor.

Severe aplastic anemia (SAA) is a rare disorder leading to bone marrow failure, which if left untreated, is invariably fatal. Conventional therapies with immunosuppressive therapy or allogeneic hematopoietic stem cell transplantation (HSCT) are highly effective. HSCT can offer a greater outcome in younger patients who have an available HLA match-related donor. Recent studies showing the addition of antithymocyte globulin (ATG) to the conditioning regimen improves engraftment and reduces the risk of graft-versus-host disease (GVHD).There are currently two ATG preparations in the USA, equine (or horse) and rabbit ATG. These agents are pharmacologically distinct, having significant differences in their pharmacokinetics and in vivo immunosuppressive effects [N Engl J Med 365(5):430-438, 2011]. Here, we report a case of two monozygotic twins with constitutional SAA that evolved to myelodysplastic syndrome (MDS) who both underwent allogeneic peripheral blood stem cell transplantation (PBSC) from the same single HLA antigen mismatched sibling donor with the only difference in the transplant regimen being the type of ATG used in the preparative regimen; one twin received horse ATG and the other received rabbit ATG during conditioning. This report emphasizes that dramatic differences in donor T cell chimerism and clinical outcomes including GVHD can occur as a consequence of the type of ATG that is utilized in the transplant conditioning regimen. These differences highlight that these agents should not be considered interchangeable drugs when used in this setting.

Oligosaccharide-mediated interactions of the envelope glycoprotein gp120 of HIV-1 that are independent of CD4 recognition.

In this study carbohydrate-mediated interactions of the envelope glycoprotein, gp120, of HIV-1 were investigated. Oligosaccharide probes (neoglycolipids), prepared from the N-glycosidically-linked chains of the natural and recombinant forms of gp120, were used in conjunction with the intact glycoprotein to investigate reactivities with a soluble carbohydrate-binding protein (lectin) known as mannose-binding protein in human serum. Evidence is presented that the high-mannose-type oligosaccharides with seven, eight and nine mannose residues from both forms of gp120 are recognized by the serum lectin, and that these reactivities are unrelated to CD4 recognition. Reactivities of the two forms of envelope glycoprotein with macrophages derived from human blood monocytes and with the mannose-specific macrophage endocytosis receptor isolated from human placental membranes were also investigated. Evidence is presented that both forms of gp120 bind to the macrophage surface by multiple interactions in addition to CD4 binding, and that among these interactions is a carbohydrate-mediated binding to the endocytosis receptor. We propose that such carbohydrate-mediated interactions could form the basis of viral attachment to a variety of healthy and diseased tissues.

Amino acid sequence of beta-galactoside-binding bovine heart lectin. Member of a novel class of vertebrate proteins.

A variety of animal tissues contain beta-galactoside-binding lectins with molecular masses in the range 13-17 kDa. There is evidence that these lectins may constitute a new protein family although their function in vivo is not yet clear. In this work the major part of the amino acid sequence of the 13 kDa lectin from bovine heart muscle has been determined. Comparison of this sequence with the cDNA-deduced sequence published for the chick embryo skin lectin showed 58% homology. Comparison of the bovine lectin sequence with partial sequences from two cDNA clones from a human hepatoma library and partial amino acid sequences of human lung lectin showed 70, 40 and 85% homology, respectively. The sequences of these vertebrate lectins are thus clearly related, supporting earlier results of immunological cross-reactivity within this group of proteins. Computer searching of protein sequence databases did not detect significant homologies between the bovine lectin sequence and other known proteins.

The preparation of DTPA-coupled antibodies radiolabeled with metallic radionuclides: an improved method.

Isotopes of iodine are often employed as radiolabels for antibodies used in radioimmunodetection studies in which tumor localization is determined by external imaging. Because of drawbacks associated with the use of these isotopes, alternative labeling methods have been considered; such as covalently attaching strong chelators so that the coupled protein may be radiolabeled with metallic radionuclides by chelation. We have developed a method of coupling the strong chelator diethylenetriaminepentaacetic acid (DTPA) which is simple, efficient, and superior to reported methods. Using the cyclic anhydride, coupling to IgG antibody is about 75% efficient and is completed in less than 1 min at neutral pH. Because the concentration of hydrolytic products is small, the coupled protein is rapidly purified for use or storage. Labeling of the protein is also accomplished rapidly and the labeled product has been shown to be stable both in vitro and in vivo.

Influence of oligosaccharide presentation on the interactions of carbohydrate sequence-specific antibodies and the selectins. Observations with biotinylated oligosaccharides.

This study was aimed at investigating the efficacy of presentation of biotinylated oligosaccharides on streptavidin-coated microwells for interactions with (a) three monoclonal antibodies directed at sialyl-Lewisa (Le(a)) or sulfo-Le(a)-related sequences, and (b) the endothelium-leukocyte adhesion molecules, the E-, L- and P-selectins which recognize both the sulfo- and sialyl-Le(a) series. With the antibodies it was observed that if the biotinylated oligosaccharide incorporated the entire antigenic determinant, and additional saccharide length was not included, the biotinyl tag spacer length was a critical factor in the strength of the binding signal. If oligosaccharide chain beyond the determinant was included, the biotinyl tag spacer length was less important. The E-selectin binding data with the biotinylated sialyl- and sulfo-oligosaccharides were in overall accord with previous knowledge. With the L- and P-selectins, however, unexpectedly low binding signals were elicited by biotinyl sulfo-Le(a) sequences relative to those with the sialyl-analogs. This suppression was more pronounced with the rodent than the human L-selectin. Such differential availabilities of oligosaccharides displayed on streptavidin may relate to biological situations, such as the differential reactivities of the three selectins with a given oligosaccharide ligand presented on different carrier proteins, or on different O-glycan cores on mucin-type glycoproteins.