Development and activation of human dendritic cells in vivo in a xenograft model of human hematopoiesis

Dendritic cells (DCs) are derived from CD34+ progenitors and play a central role in the development of immune responses and in tolerance. Their therapeutic potential underscores the need for in vivo models that accurately recapitulate human DC development and function to provide a better understanding of DC biology in health and disease. Using nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice transplanted with human CD34+ cells as a model of human hematopoiesis, we examined DC ontogeny. Progenitors of both myeloid (m) and plasmacytoid (p) DCs were identified in the bone marrow of mice up to 24 weeks after transplant, indicating ongoing and sustained production of DCs after initial engraftment. To determine whether human DCs derived from transplanted stem cells were functional, their response to acute inflammation using lipopolysaccharide (LPS) was examined. Eighteen hours after LPS administration, a dramatic increase in the plasma levels of the human inflammatory cytokines interleukin (IL)-8, IL-10, tumor necrosis factor-alpha, and IL-12p70 was observed. Only mDCs and not pDCs responded in vivo to LPS by upregulating CD86 and CD83. In vivo activation of human mDCs resulted in a substantial increase in the ability of mDCs to induce the proliferation of naive human T cells. Taken together, these data indicate that human CD34+ cells seem to have differentiated appropriately within the NOD/SCID microenvironment into DCs that are developmentally, phenotypically, and functionally similar to the DC subsets found in humans.     

Allotypes in Basilea rabbits

Basilea rabbits produce immunoglobulin molecules, practically all of which have lambda light chains rather than kappa chains. This variant strain was derived form a homozygous (b9/b9) male. Sensitive serological methods revealed that at least some homozygous bas/bas individuals possess traces (about 100 ng/ml) of b9 molecules. This level usually increases to almost 1 microgram/ml upon hyperimmunization with pneumococcal or streptococcal vaccines. One exceptional rabbit, with 50 micrograms/ml of b9 molecules, was found. In spite of the presence of b9 molecules in early pre-immune bleeds, homozygous bas/bas rabbits produce strong anti-b9 antibodies; i.e., they are capable of making autoantibodies. These anti-b9 allotypic antisera were not distinguishable by our methods from routinely produced anti-b9.     

The role of the microtubules in tumor necrosis factor-alpha-induced endothelial cell permeability

Tumor necrosis factor (TNF)-alpha, a major proinflammatory cytokine, triggers endothelial cell activation and barrier dysfunction which are implicated in the pathogenesis of pulmonary edema associated with acute lung injury syndromes. The mechanisms of TNF-alpha-induced vascular permeability are not completely understood. Our initial experiments demonstrated that TNF-alpha-induced decreases in transendothelial electrical resistance across human pulmonary artery endothelial cells are independent of myosin light chain phosphorylation catalyzed by either myosin light chain kinase or Rho kinase. We next assessed the involvement of another cytoskeletal component, the tubulin-based microtubule network, and found TNF-alpha to induce a decrease in stable tubulin content and partial dissolution of peripheral microtubule network as evidenced by anti-acetylated tubulin and anti-beta-tubulin immunofluorescent staining, respectively. Microtubule-stabilizing agents, paclitaxel and epothilone B, significantly attenuated TNF-alpha-induced decreases in transendothelial electrical resistance, inhibited the cytokine-induced increases in actin stress fibers, formation of intercellular gap, and restored the TNF-alpha-compromised vascular endothelial (VE)-cadherin-based cell-cell junctions. Importantly, neither TNF-alpha nor paclitaxel treatment was associated with endothelial cell apoptosis. Inhibition of p38 mitogen-activated protein kinase by SB203580 significantly attenuated TNF-alpha-induced microtubule destabilization, actin rearrangement, and endothelial barrier dysfunction. These results strongly suggest the involvement of microtubule rearrangement in TNF-alpha-induced endothelial cell permeability via p38 mitogen-activated protein kinase activation.     

Quantitation of IgE on the basophil surface in atopic patient

A radioimmunoassay was employed in order to quantitate IgE on the basophil surface in 6 normal and 10 pollen sensitive subjects. A basophil-rich fraction obtained from whole blood in a Ficoll gradient was incubated with specific anti-IgE; the inhibition produced by surface IgE was standard inhibition curve. Atopic patients had on the average about 10-30 times as many IgE molecules per basophil as control subjects. Total and specific IgE as well as degranulation by Lollium perenne crude extract were also studied in both populations and their results compared with those of basophil IgE quantitation.     

Fine-needle aspiration diagnosis of Kaposi's sarcoma in a developing country

Fine-needle aspiration (FNA) cytology was performed on 15 patients with peripheral lymphadenopathy and/or skin lesions referred to the Department of Pathology of the Hospital Central of Maputo, Maputo, Mozambique. Epitrochlear lymph nodes were the most frequently aspirated site. All aspirates allowed diagnoses of Kaposi's sarcoma (KS). Smears contained loosely cohesive clusters of bland spindle cells, with a radial arrangement and nuclear crush artifacts. These diagnostic clues have not been described in other spindle-cell intranodal lesions that should be considered in differential diagnoses. Taking into consideration the high prevalence of AIDS and limited resources for diagnosis in Africa, FNA cytology appears to be a useful method for the diagnosis of KS in developing countries, reducing the necessity for surgical lymph node excision.     

IS1245 Restriction Fragment Length Polymorphism Typing of Mycobacterium avium Isolates: Proposal for Standardization

Mycobacterium avium has become a major human pathogen, primarily due to the emergence of the AIDS epidemic. Restriction fragment length polymorphism (RFLP) typing, using insertion sequence IS1245 as a probe, provides a powerful tool in the molecular epidemiology of M. avium-related infections and will facilitate well-founded studies into the sources of M. avium infections in animal and environmental reservoirs. The standardization of this technique allows computerization of IS1245 RFLP patterns for comparison on a local level and the establishment of M. avium DNA fingerprint databases for interlaboratory comparison. Moreover, by combining international DNA typing results of M. avium complex isolates from a broad spectrum of sources, long-lasting questions on the epidemiology of this major agent of mycobacterial infections will be answered.     

Identification and typing of herpes simplex viruses with monoclonal antibodies.

Monoclonal antibodies which reacted with type-specific antigens of herpes simplex virus type 2 or with antigens shared by herpes simplex virus types 1 and 2 were used in an indirect immunofluorescence assay to type virus isolates and to detect viral antigens in cells obtained from herpetic lesions. Complete concordance was obtained for 42 isolates typed by endonuclease restriction analysis of viral DNA and by indirect immunofluorescence with monoclonal antibodies. Examination of a limited number of ulcerative lesions revealed that indirect immunofluorescence and virus isolation were comparable in detecting herpes simplex virus. The results indicate that monoclonal antibodies can be used to accurately identify and type isolates of herpes simplex virus.     

Identification of MEN1 gene mutations in sporadic carcinoid tumors of the lung

Lung carcinoids occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). There are no well defined genetic abnormalities known to occur in these tumors. We studied 11 sporadic lung carcinoids for loss of heterozygosity (LOH) at the locus of the MEN1 gene on chromosome 11q13, and for mutations of the MEN1 gene using dideoxy fingerprinting. Additionally, a lung carcinoid from a MEN1 patient was studied. In four of 11 (36%) sporadic tumors, both copies of the MEN1 gene were inactivated. All four tumors showed the presence of a MEN1 gene mutation and loss of the other allele. Observed mutations included a 1 bp insertion, a 1 bp deletion, a 13 bp deletion and a single nucleotide substitution affecting a donor splice site. Each mutation predicts truncation or potentially complete loss of menin. The remaining seven tumors showed neither the presence of a MEN1 gene mutation nor 11q13 LOH. The tumor from the MEN1 patient showed LOH at chromosome 11q13 and a complex germline MEN1 gene mutation. The data implicate the MEN1 gene in the pathogenesis of sporadic lung carcinoids, representing the first defined genetic alteration in these tumors.