Molecular and spatial epidemiology of cryptosporidiosis in children in a semiurban community in South India

Cryptosporidium spp. are a leading cause of diarrhea in Indian children, but there are no data for prevalent species or subgenotypes. Genetic characterization of Cryptosporidium spp. by PCR-restriction fragment length polymorphism and spatial analysis of cases using Geographical Information Systems technology was carried out for 53 children with cryptosporidial diarrhea in an urban slum. The two most common species were C. hominis (81%) and C. parvum (12%). Other species identified were C. felis and C. parvum (mouse genotype). Five subgenotypes were identified at the Cpgp40/15 locus. Subgenotype Ia predominated among C. hominis isolates, and all C. parvum isolates were subgenotype Ic. C. hominis infection was associated with a greater severity of diarrhea. Sequencing of the Cpgp40/15 alleles of C. felis and C. parvum (mouse genotype) revealed similarities to subgenotype IIa and C. meleagridis, respectively. Space-time analysis revealed two clusters of infection due to C. hominis Ia, with a peak in February 2005. This is the first study to demonstrate space-time clustering of a single subgenotype of C. hominis in a setting where cryptosporidiosis is endemic. Molecular characterization and spatial analysis have the potential to further the understanding of disease and transmission in the community.     

Molecular characterization of G11P[25] and G3P[3] human rotavirus strains associated with asymptomatic infection in South India

Rotaviruses are the major etiological agents of diarrhea in children less than 5 years of age. Two unusual rotavirus strains not previously reported in India, G11P[25] (CRI 10795) and G3P[3] (CRI 33594) were isolated from faecal samples of asymptomatic children in India. The strains were characterized by sequence analysis of the genes encoding the VP7, VP4, VP6, and NSP4. The G11P[25] strain was closely related to the human G11P[25] strains from Bangladesh (with 98% identity at the nucleotide [nt] level and the amino acid [aa] level for the VP7 gene and 96% identity at the nt and 98% at the aa level for the VP4 gene). The G3P[3] strain was found to be related to a G3P[3] strain isolated in Thailand (CMH222; 88% identity at the nt level and 97% at aa level for the VP7 gene and 84% identity at the nt level and 90% at the aa level for the VP4 gene). Phylogenetic analysis of the VP6 and the NSP4 genes revealed that the Vellore G11P[25] strain was of VP6 subgroup II and NSP4 genotype B. The G3P[3] strain was identified as NSP4 genotype C and the VP6 gene showed 97% identity at the deduced amino acid level with strain CMH222 (Thailand) strain but did not cluster with sequences of SGI, SGII, SGI+II or SG-nonI/nonII. Both strains had gene segments of animal rotavirus origin suggesting inter-species transmission of rotavirus, and in the case of G11P[25] possibly underwent reassortment subsequently with human strains resulting in an animal-human hybrid strain.     

Aplastic anemia and paroxysmal nocturnal hemoglobinuria: search for a pathogenetic link

The association of paroxysmal nocturnal hemoglobinuria (PNH) and aplastic anemia (AA) raises the yet unresolved questions as to whether these two disorders are different forms of the same disease. We compared two groups of patients with respect to cytogenetic features, glycosylphosphatidylinositol (GPI)-linked protein expression, protein C/protein S/thrombomodulin/antithrombin III activity, and PIG-A gene expression. The first group consisted of eight patients with PNH (defined as positive Ham and sucrose tests at diagnosis), and the second, 37 patients with AA. Twelve patients with AA later developed a PNH clone. Monoclonal antibodies used to study GPI-linked protein expression (CD14 [on monocytes], CD16 [on neutrophils], CD48 [on lymphocytes and monocytes], CD67 [on neutrophils and eosinophils], and, more recently, CD55, CD58, and CD59 [on erythrocytes]) were also tested on a cohort of 20 normal subjects and five patients with constitutional AA. Ham and sucrose tests were performed on the same day as flow- cytometric analysis. Six of 12 patients with AA, who secondarily developed a PNH clone, had clinical symptoms, while all eight patients with PNH had pancytopenia and/or thrombosis and/or hemolytic anemia. Cytogenetic features were normal in all but two patients. Proteins C and S, thrombomodulin, and antithrombin III levels were within the normal range in patients with PNH and in those with AA (with or without a PNH clone). In patients with PNH, CD16 and CD67 expression were deficient in 78% to 98% of the cells and CD14 in 76% to 100%. By comparison, a GPI-linked defect was detected in 13 patients with AA, affecting a mean of 32% and 33% of CD16/CD67 and CD14 cell populations, respectively. Two of three tested patients with PNH and 1 of 12 patients with AA had a defect in the CD48 lymphocyte population. In a follow-up study of our patient cohort, we used the GPI-linked molecules on granulocytes and monocytes investigated earlier and added the study of CD55, CD58, and CD59 on erythrocytes. Two patients with PNH and 14 with AA were studied for 6 to 13 months after the initial study. Among patients with AA, four in whom no GPI-anchoring defect was detected in the first study had no defect in follow-up studies of all blood-cell subsets (including erythrocytes). Analysis of granulocytes, monocytes, and erythrocytes was performed in 7 of 13 AA patients in whom affected monocytes and granulocytes were previously detected. A GPI-anchoring defect was detected on erythrocytes in five of six.(ABSTRACT TRUNCATED AT 400 WORDS)     

Failure-free survival in a prospective cohort of patients with chronic graft-versus-host disease

Failure-free survival, defined as the absence of relapse, non-relapse mortality or addition of another systemic therapy, has been proposed as a potential endpoint for clinical trials, but its use has only been reported for single-center studies. We measured failure-free survival in a prospective observational cohort of patients (n=575) with both newly diagnosed and existing chronic graft-versus-host disease from nine centers. Failure was observed in 389 (68%) patients during the observation period. The median follow up of all patients was 30.9 months, and the median failure-free survival was 9.8 months (63% at 6 months, 45% at 1 year, and 29% at 2 years). Of the variables measured at enrollment, ten were associated with shorter failure-free survival: higher National Institutes of Health 0–3 skin score, higher National Institutes of Health 0–3 gastrointestinal score, worse range of motion summary score, lower forced vital capacity (%), bronchiolitis obliterans syndrome, worse quality of life, moderate to severe hepatic dysfunction, absence of treatment for gastric acid, female donor for male recipient, and prior grade II–IV acute graft-versus-host disease. Addition of a new systemic treatment, the major cause of failure, was associated with an increased risk of subsequent non-relapse mortality (hazard ratio=2.06, 95% confidence interval: 1.29–3.32; P<0.003) and decreased survival (hazard ratio=1.51, 95% confidence interval: 1.04–2.18; P<0.03). These results show that fewer than half of patients on systemic treatment will be failure-free survivors at 1 year, and fewer than a third will reach 2 years without experiencing failure. Better treatments are needed for chronic graft-versus-host disease. Clinicaltrials.gov identifier: {"type":"clinical-trial","attrs":{"text":"NCT00637689","term_id":"NCT00637689"}}NCT00637689.     

Stratifying risk of recurrence in stage II colorectal cancer using deregulated stromal and epithelial microRNAs

MicroRNAs (miRNAs) enable colonic epithelial cells to acquire malignant characteristics and metastatic capabilities. Recently, cancer relevant miRNAs deregulated during disease progression have also been identified in tumor-associated stroma.By combining laser-microdissection (LMD) with high-throughput screening and high-sensitivity quantitation techniques, miRNA expression in colorectal cancer (CRC) specimens and paired normal colonic tissue was independently characterized in stromal and epithelial tissue compartments. Notably, deregulation of the key oncogene miR-21 was identified exclusively as a stromal phenomenon and miR-106a, an epithelial phenomenon in the malignant state.MiRNAs identified in this study successfully distinguished CRC from normal tissue and metastatic from non-metastatic tumor specimens. Furthermore, in a separate cohort of 50 consecutive patients with CRC, stromal miR-21 and miR-556 and epithelial miR-106a expression predicted short disease free survival (DFS) and overall survival (OS) in stage II disease: miR-21 (DFS: HR = 2.68, p = 0.015; OS: HR = 2.47, p = 0.029); miR-556 (DFS: HR = 2.60, p = 0.018); miR-106a (DFS: HR = 2.91, p = 0.008; OS: HR = 2.25, p = 0.049); combined (All High vs. All Low. DFS: HR = 5.83, p = 0.002; OS: HR = 4.13, p = 0.007).These data support the notion that stromal as well as epithelial miRNAs play important roles during disease progression, and that mapping patterns of deregulated gene expression to the appropriate tumor strata may be a valuable aid to therapeutic decision making in CRC.     

Epstein-Barr virus lymphoproliferation after bone marrow transplantation

We review 15 cases of secondary B-cell lymphoproliferative disorders that occurred among 2,475 patients who received allogeneic bone marrow transplants (BMTs) at the Fred Hutchinson Cancer Research Center (Seattle) between 1969 and 1987. The histopathologic findings in 14 of the 15 patients spanned a wide spectrum of lymphoproliferative lesions. One patient had features characteristic of angioimmunoblastic lymphadenopathy. Epstein-Barr virus (EBV) genomic sequences were identified by Southern blot analysis in each of the 13 patients evaluated. Ten of the 12 lesions evaluated originated in donor cells. In two patients, who had mixed chimerism after transplantation, the lesions originated in host cells. The combined evidence from immunoglobulin light chain staining and the analysis of immunoglobulin heavy chain gene rearrangement indicated that the lesions in most patients represented polyclonal proliferations that gave rise to clonal subpopulations. The results indicate an overall actuarial incidence of 0.6% for this complication in BMT recipients. Anti-CD3 monoclonal antibody (MoAb) treatment of acute graft-v-host disease (GVHD) and T cell depletion of the donor marrow were statistically significant risk factors, and GVHD appeared to play a contributing role, particularly in the setting of human leukocyte antigen (HLA) disparity. Two patients had no identifiable risk factors. Prophylaxis or treatment with acyclovir had no detectable effect in the patients; all but two died with uncontrolled lymphoproliferation.     

Monoclonal antibody BA-1 does not bind to hematopoietic precursor cells

Monoclonal antibody BA-1 binds to B lymphocytes, to cells from most cases of non-T acute lymphoblastic leukemia (ALL), and weakly to neutrophils. To determine whether BA-1 also reacts with hematopoietic progenitor cells (HPC), we studied the effect of removal of BA-1+ cells from human bone marrow on the proliferation in vitro of the trilineage precursor cell CFU-GEMM, and on the committed progenitor cells of granulopoiesis (CFU-C) and erythropoiesis (BFU-E/CFU-E). Complement- mediated cytotoxicity using BA-1 at concentrations far beyond those required to lyse BA-1+ bone marrow cells and ALL cells did not result in inhibition of colony formation in any of the assays. A rosette separation method, using ox red blood cells coated with BA-1, resulted in enrichment of HPC in the BA-1-depleted interface, whereas very few HPC were found in the BA-1-enriched pellet. Both methods indicate that BA-1 does not bind to HPC, although binding of the antibody to the lymphohematopoietic stem cell cannot be excluded yet. The high cytotoxic capacity of the IgM antibody BA-1, and the lack of reactivity with HPC, make the antibody particularly suitable for use in autologous bone marrow transplantation for patients with ALL.