An efficient SNP system for mouse genome scanning and elucidating strain relationships

A set of 1638 informative SNP markers easily assayed by the Amplifluor genotyping system were tested in 102 mouse strains, including the majority of the common and wild-derived inbred strains available from The Jackson Laboratory. Selected from publicly available databases, the markers are on average ~1.5 Mb apart and, whenever possible, represent the rare allele in at least two strains. Amplifluor assays were developed for each marker and performed on two independent DNA samples from each strain. The mean number of polymorphisms between strains was 608±136 SD. Several tests indicate that the markers provide an effective system for performing genome scans and quantitative trait loci analyses in all but the most closely related strains. Additionally, the markers revealed several subtle differences between closely related mouse strains, including the groups of several 129, BALB, C3H, C57, and DBA strains, and a group of wild-derived inbred strains representing several Mus musculus subspecies. Applying a neighbor-joining method to the data, we constructed a mouse strain family tree, which in most cases confirmed existing genealogies.     

Tolerance in mixed chimerism – a role for regulatory cells?

The establishment of mixed hematopoietic chimerism induces life-long donor-specific organ graft tolerance while obviating the need for chronic immunosuppression. Recent advances have dramatically reduced the conditioning toxicity required to achieve mixed chimerism. We argue that the achievement of high levels of donor chimerism ensures life-long deletion of donor-reactive T cells, precluding and obviating the need for regulatory mechanisms in the maintenance of tolerance. However, in situations where high levels of donor chimerism cannot be established or sustained, control of immune responsiveness can be achieved through additional mechanisms, including regulatory T cells.     

Characterization of Anticapsular Monoclonal Antibodies That Regulate Activation of the Complement System by the Cryptococcus neoformans Capsule

Incubation of the encapsulated yeast Cryptococcus neoformans in human serum leads to alternative pathway-mediated deposition of C3 fragments in the capsule. We examined the ability of monoclonal antibodies (MAbs) specific for different epitopes of the major capsular polysaccharide to alter the kinetics for classical and alternative pathway-mediated deposition of C3 onto a serotype A strain. We studied MAbs reactive with capsular serotypes A, B, C, and D (MAb group II); serotypes A, B, and D (MAb group III); and serotypes A and D (MAb group IV). The MAb groupings are based on antibody variable region usage which determines the antibody molecular structure. When both the classical and alternative pathways were operative, group II MAbs induced early classical pathway-mediated binding of C3 but reduced the overall rate of C3 accumulation and the amount of bound C3. Group III MAbs closely mimicked the effects of group II MAbs but exhibited reduced support of early classical pathway-facilitated accumulation of C3. Depending on the antibody isotype, group IV MAbs slightly or markedly enhanced early binding of C3 but had no effect on either the rate of C3 accumulation or the amount of bound C3. When the classical pathway was blocked, group II and III MAbs markedly suppressed C3 binding that normally would have occurred via the alternative pathway. In contrast, MAbs of group IV had no effect on alternative pathway-mediated C3 binding. These results indicate that anticapsular antibodies with different epitope specificities may have distinct regulatory effects on activation and binding of C3.Cryptococcus neoformans is the etiological agent of cryptococcal meningitis, a life-threatening infection of particular importance in patients with deficiencies in cellular immunity, most notably patients with the AIDS. The yeast is surrounded by a polysaccharide capsule that is composed primarily of glucuronoxylomannan (GXM), which has a linear (1→3)-α-d-mannopyranan backbone bearing β-d-xylopyranosyl, β-d-glucopyranosyluronic acid, and O-acetyl substituents (3, 9, 54). The cryptococcal capsule occurs as four major serotypes (A, B, C, and D) and is an essential virulence factor for the yeast.One of the most striking features of the cryptococcal capsule is its ability to activate the alternative complement pathway. Incubation of encapsulated cryptococci in normal human serum (NHS) leads to the deposition of 10⁷ to 10⁸ C3 fragments on the yeast (28, 56). The C3 is deposited at the surface and throughout the capsule (30). Available evidence indicates that the amount of anti-GXM antibodies found in NHS is not sufficient to initiate the classical pathway (24); consequently, activation and binding of C3 to the cryptococcal capsule are mediated entirely by the alternative complement pathway (29, 30, 55). One of the hallmark features of alternative pathway deposition of C3 onto encapsulated cryptococci is a delay of 5 to 8 min before readily detectable amounts of C3 are found on yeast cells incubated in NHS (29, 55). Once past the initial lag, C3 fragments rapidly accumulate on the yeast cells as incubation proceeds for an additional 10 min.Recently, there has been interest in antibody-mediated resistance to cryptococcosis. Monoclonal antibodies (MAbs) have been proposed for treatment of cryptococcosis (7), and immunization with GXM-protein conjugates has been suggested for prevention of cryptococcosis (6, 12, 13). However, it is becoming increasingly clear that anti-GXM MAbs may have distinct specificities and biological activities. Anti-GXM MAbs which differ in (i) reactivities with GXM of the four major serotypes (2), (ii) apparent binding sites in the cryptococcal capsule (32, 37), and (iii) abilities to provide protection in a murine model of cryptococcosis (32, 37) have been described. Some differences in biological activity are related to differences in the epitope specificities of the various MAbs (32, 37).One means by which antibodies could enhance resistance to cryptococcosis is through accelerated deposition of opsonic C3 fragments via the action of the classical pathway. Such an acceleration would reduce or eliminate the 5- to 8-min lag that occurs during alternative pathway-mediated deposition of C3 fragments. The objectives of our study were to evaluate the effects of anti-GXM MAbs on the kinetics and sites for deposition of C3 fragments into the cryptococcal capsule. We examined several well-characterized antibodies that differed in the epitope specificity of the MAbs. The results showed that MAbs with different isotypes and epitope specificities had distinctly different effects on activation and binding of C3 via the classical and alternative pathways; many antibodies markedly suppressed C3 binding, some antibodies accelerated C3 binding, and other antibodies had little or no effect.     

Heminested inverse PCR for IS6110 fingerprinting of Mycobacterium tuberculosis strains.

A heminested inverse PCR (HIP) for the amplification of sequences flanking the Mycobacterium tuberculosis insertion sequence IS6110 has been developed. The method depends upon primers that anneal to IS6110 at sites between its 5' end and the closest BsrFI site. The accuracy of HIP was demonstrated by the amplification of sequences within plasmid constructs carrying one or two copies of the insertion sequence IS986 in different orientations. The identities of the amplicons produced from strains carrying a single copy of IS6110 were verified by nucleotide sequencing. Analyses of 204 M. tuberculosis strains including those involved in outbreaks showed that IS6110 HIP is highly discriminatory and reproducible. HIP fingerprinting of these 204 strains generated 136 distinct types, and its discriminatory power was equivalent to that of standard restriction fragment length polymorphism analysis. The method is therefore of value for the rapid fingerprinting of M. tuberculosis strains for epidemiological purposes.     

Phenotypic expansion of Bosch–Boonstra–Schaaf optic atrophy syndrome and further evidence for genotype–phenotype correlations

Bosch–Boonstra–Schaaf Optic Atrophy Syndrome (BBSOAS) is an autosomal dominant neurodevelopmental disorder caused by loss‐of‐function variants in NR2F1 and characterized by visual impairment, developmental delay, and intellectual disability. Here we report 18 new cases, provide additional clinical information for 9 previously reported individuals, and review an additional 27 published cases to present a total of 54 patients. Among these are 22 individuals with point mutations or in‐frame deletions in the DNA‐binding domain (DBD), and 32 individuals with other types of variants including whole‐gene deletions, nonsense and frameshift variants, and point mutations outside the DBD. We corroborate previously described clinical characteristics including developmental delay, intellectual disability, autism spectrum disorder diagnoses/features thereof, cognitive/behavioral anomalies, hypotonia, feeding difficulties, abnormal brain MRI findings, and seizures. We also confirm a vision phenotype that includes optic nerve hypoplasia, optic atrophy, and cortical visual impairment. Additionally, we expand the vision phenotype to include alacrima and manifest latent nystagmus (fusional maldevelopment), and we broaden the behavioral phenotypic spectrum to include a love of music, an unusually good long‐term memory, sleep difficulties, a high pain tolerance, and touch sensitivity. Furthermore, we provide additional evidence for genotype–phenotype correlations, specifically supporting a more severe phenotype associated with DBD variants.     

Nonmyeloablative bone marrow transplantation: Infectious complications in 65 recipients of HLA-identical and mismatched transplants.

Infections are a common complication of allogeneic bone marrow transplantation and the leading cause of transplantation-related mortality. It had been hypothesized that transplantation following nonmyeloablative preparative regimens would result in fewer infections by causing less mucosal injury, less graft-versus-host disease, and allowing earlier immune reconstitution. We have retrospectively reviewed the infectious complications of 65 consecutive patients with advanced hematologic malignancies who underwent bone marrow transplantation using a novel preparative regimen consisting of cyclophosphamide, thymic irradiation, and in vivo T-cell depletion. Cytomegalovirus (CMV) infection occurred in 52% of cases in which the donor or recipient had evidence of prior CMV exposure. Using a strategy of preemptive therapy and secondary prophylaxis with ganciclovir, no CMV disease occurred. Infections with gram-positive bacteria predominated over the first 100 days after bone marrow transplantation. Thereafter, the relative proportion of gram-negative infections increased without a significant increase in episodes of neutropenia. The rate of bacterial infections was not influenced by relapse of the underlying malignancy. Seven patients developed infections with Aspergillus species, which was the most common infectious cause of death in these patients. Infections with viruses other than CMV (n=10) and with protozoan organisms (n=2) also occurred. The use of HLA-mismatched donors, the occurrence of grade II-IV acute graft-versus-host disease, and treatment with corticosteroids did not influence the risk of CMV or bacterial or fungal infections in patients who underwent transplantation following this preparative regimen. Overall, the incidence and spectrum of infections in this series was similar to the reported incidence of infections following conventional myeloablative allogeneic stem cell transplantation. We conclude that a quantitative T-cell deficiency in these extensively T-cell depleted patients may be a risk factor for infection, even in the absence of graft-versus-host disease.     

A method for real-time, evidence-based general medical attending rounds.

PURPOSE: To assess the utility and practicality of an evidence-based format in internal medicine attending rounds. METHOD: Two randomly selected teams of residents and medical students in the internal medicine program at the Montefiore Medical Center participated in "Evidence-Based Medicine Attending Month." The process entailed the development of patient-based, searchable questions, a search for the evidence, the critical appraisal of the retrieved literature, and the application of the evidence to the care of the patient. At the last meeting, participants evaluated each case by answering three questions about whether the process (1) had changed the medical management of the patient during the admission, (2) had changed the way they would manage similar patients in the future, and (3) had informed them about the disease process in general. RESULTS: A total of 12 of 16 formal EBM questions were developed and assessed (75% completion rate) during the four-week period, in addition to the standard background literature reviews usually performed. Twenty-two articles were retrieved and critically appraised. The evaluation demonstrated that 50% of the participants felt the process had changed the active management of patients currently treated by the team, 75% reported that the process would affect the care of future patients with comparable medical problems, and over 90% believed the program had informed them about the disease process. CONCLUSIONS: The formal EBM approach was conveniently implemented and enhanced the learning experience of the participants. It helped inform students' and residents' patient care at the time and their attitudes towards future patients. Hence, it is both practical and useful to perform formal EBM attending rounds.     

Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis isolates recovered from wild animal species

Mycobacterial isolates were obtained by radiometric culture from 33 different species of captive or free-ranging animals (n = 106) and environmental sources (n = 3) from six geographic zones within the United States. The identities of all 109 isolates were confirmed by using mycobactin J dependence and characterization of five well-defined molecular markers, including two integration loci of IS900 (loci L1 and L9), one Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis)-specific sequence (locus 251), and one M. avium subsp. avium-specific marker (IS1245), as well as hsp65 and IS1311 restriction endonuclease analyses. Seventy-six acid-fast isolates were identified as M. paratuberculosis, 15 were identified as belonging to the M. avium-M. intracellulare complex (but not M. paratuberculosis), and the remaining 18 were identified as mycobacteria outside the M. avium-M. intracellulare complex. Fingerprinting by multiplex PCR for IS900 integration loci clustered 67 of the 76 M. paratuberculosis strains into a single clade (designated clade A18) and had a Simpson's diversity index (D) of 0.53. In contrast, sequence-based characterization of a recently identified M. paratuberculosis short sequence repeat (SSR) region enabled the differentiation of the M. paratuberculosis isolates in clade A18 into seven distinct alleles (D = 0.75). The analysis revealed eight subtypes among the 33 species of animals, suggesting the interspecies transmission of specific strains. Taken together, the results of our analyses demonstrate that SSR analysis enables the genetic characterization of M. paratuberculosis isolates from different host species and provide evidence for the host specificity of some M. paratuberculosis strains as well as sharing of strains between wild and domesticated animal species.