Suggested new breakpoints of anti-MERS-CoV antibody ELISA titers: performance analysis of serologic tests.

To provide optimal cut-off values of anti-Middle East respiratory syndrome coronavirus (MERS-CoV) serologic tests, we evaluated performance of ELISA IgG, ELISA IgA, IFA IgM, and IFA IgG using 138 serum samples of 49 MERS-CoV-infected patients and 219 serum samples of 219 rRT-PCR-negative MERS-CoV-exposed healthcare personnel and patients. The performance analysis was conducted for two different purposes: (1) prediction of neutralization activity in MERS-CoV-infected patients, and (2) epidemiologic surveillance of MERS-CoV infections among MERS-CoV-exposed individuals. To evaluate performance according to serum collection time, we used 'days post onset of illness (dpoi)' and 'days post exposure (dpex)' assessing neutralization activity and infection diagnosis, respectively. Performance of serologic tests improved with delayed sampling time, being maximized after a seroconversion period. In predicting neutralization activity, ELISA IgG tests showed optimal performance using sera collected after 21 dpoi at cut-off values of OD ratio 0.4 (sensitivity 100% and specificity 100%), and ELISA IgA showed optimal performance using sera collected after 14 dpoi at cut-off value of OD ratio 0.2 (sensitivity 85.2% and specificity 100%). In diagnosis of MERS-CoV infection, ELISA IgG exhibited optimal performance using sera collected after 28 dpex, at a cut-off value of OD ratio 0.2 (sensitivity 97.3% and specificity 92.9%). These new breakpoints are markedly lower than previously suggested values (ELISA IgG OD ratio 1.1, sensitivity 34.8% and specificity 100% in the present data set), and the performance data help serologic tests to be practically used in the field of MERS management.

DC-SIGN-specific liposomal targeting and selective intracellular compound delivery to human myeloid dendritic cells: implications for HIV disease.

Myeloid dendritic cells (MyDCs), prime stimulators of antigen-specific immunity, can serve as one of the major reservoirs for human immunodeficiency virus type-1 (HIV-1). Utilizing mature monocyte-derived MyDCs generated with granulocyte/macrophage colony-stimulating factor, interleukin-4, and tumour necrosis factor-alpha as an in vitro model, we here present the first proof of concept for liposomal compound delivery to these cells by specifically addressing CD209, i.e. DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), a MyDC-associated C-type lectin implicated in the transmission of HIV-1 to T helper cells. By employing a liposomally entrapped tracer, calcein, we demonstrate by flow cytometry and mathematics a superior targeting efficacy for DC-SIGN, as compared with select other MyDC markers (CD1a, CD4, CD45R0, and CD83). Fluorescence microscopy reveals time-dependent surface binding and intracellular uptake of DC-SIGN-specific liposomes by both immature and mature MyDCs. This pilot study implies that liposomal targeting to CD209 and related C-type lectins may afford therapeutic intracellular drug delivery to MyDCs and other reservoir and nonreservoir cells susceptible to infection with HIV-1.

Megakaryothrombopoiesis during ex vivo expansion of human cord blood CD34+ cells using thrombopoietin.

Thrombopoietin (TPO) is one of the most promising stimulants for ex vivo expansion of haematopoietic stem cells. Previously, we have found that TPO induces a characteristic pattern of apoptosis during ex vivo expansion of human cord blood (CB) CD34+ cells and that the TPO-induced apoptotic cells belong to megakaryocyte (MK) lineage. In this study, we have examined the maturation of MK and platelet production in association with the TPO-induced apoptosis. CD34+ cells, purified from human CB, were expanded in serum-free conditions stimulated with TPO. Apoptosis was confirmed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL) assay and electron microscopy (EM). Simultaneous measurement of DNA content and immunophenotyping revealed that the cells with higher DNA content (>8 N) constituted less than 5% of the CD41+ fractions until day 14, implying premature apoptosis of MKs before full polyploidization. Nevertheless, EM observation showed not only platelet territories but also newly produced platelets in which granules and microfilaments could be identified. Furthermore, flow cytometry demonstrated that the platelet fraction expressed P-selectin and an activation motif on GPIIb/IIIa recognized by monoclonal antibody PAC-1 upon stimulation with adenosine diphosphate (ADP). In addition, periodic acid-Schiff (PAS)-positive materials and nonspecific esterase activities could be demonstrated. Therefore, it is suggested that platelet production and the accompanying processes, rather than apoptosis only, be hastened during the ex vivo expansion of CB CD34+ cells when using TPO.

Development of a new epitope tag recognized by a monoclonal antibody to Rickettsia typhi.

The epitope recognized by a mouse monoclonal antibody (MAb) to the crystalline surface layer protein of Rickettsia typhi, SRT10, was mapped to 10 amino acid residues (SRTag TFIGAIATDT). The oligonucleotide sequence covering the epitope recognized by SRT10 was inserted into a mammalian expression vector together with multiple cloning sites. When the SRTag was fused in frame to the coding region of the NCC27/CLIC1 gene and expressed in mammalian cells, the MAb SRT10 could detect the tagged protein by immunoblotting, immunocytochemistry, and immunoprecipitation. In addition to the SRT-NCC27/CLIC1, SRT10 could detect N-terminal-tagged MEF2D and C-terminal-tagged CD4 by immunocytochemistry. We suggest that this specific recognition of the SRTag by SRT10 is generally applicable to cellular and molecular biology research that requires the expression and detection of fusion proteins.

Cell cycling status of human cord blood CD34+ cells during ex vivo expansion is related to the level of very late antigen expression.

Very late antigen-4 (VLA-4), which binds to the extracellular matrix protein fibronectin, is an integrin molecule known to be modulated during mobilization of CD34+ cells, and to be involved in signaling the mobilization stimuli. On the hypothesis that cell cycling status might be different depending on the level of VLA-4 expression, we investigated the DNA contents of human cord blood CD34+ cells during ex vivo expansion by recombinant human thrombopoietin and flt3-ligand with simultaneous measurement of surface VLA-4 at the 1st and 4th week. During this ex vivo expansion, expression of VLA-4 increased and almost all cells became VLA-4+ until the 4th day of culture. Expression of VLA-4 was maintained in the major population of the cultured cells until the 4th week. The cells in S/G2/M phase were greater in number in VLA-4 high fraction than in VLA-4 low fraction (n=4, p<.001). Furthermore, the fraction of cells in S/G2/M phase increased as the expression of VLA-4 became higher. These results suggest that cord blood CD34+ cells expressing high levels of VLA-4 have more proliferative activities.

Cloning and sequencing of the gene encoding the candidate HtrA of Rickettsia typhi.

We screened a phage library of Rickettsia typhi with a polyclonal antiserum to clone genes which encode immunogenic proteins of R. typhi. Among several clones obtained, one clone codes for a 466-amino-acid protein similar to the heat-shock protein, HtrA. The deduced rickettsial HtrA contains a putative signal peptide sequence at the N-terminus, a serine protease-like domain, and two PDZ domains. The recombinant protein of rickettsial HtrA reacted with sera from patients with murine typhus and tsutsugamushi disease. We suggest that this gene and its recombinant protein would be valuable for the immunologic diagnosis of rickettsial diseases.

Differential activation of MAP kinase family members triggered by CD99 engagement.

The molecular basis for the modulatory properties of CD99 is not well understood. Treatment of human Jurkat T lymphocytes with anti-CD99 antibody led to activation of three mitogen-activated protein kinase (MAPK) members, ERK, JNK, and p38 MAPK, along with homotypic aggregation. While phosphorylation of ERK and JNK was inhibited by the pretreatment of a PKC inhibitor, bisindolylmaleimide I, activation of p38 MAPK was upregulated by the same pretreatment. The signaling pathways to MAPKs by CD99 engagement were independent of PI-3 kinase, distinguishing from those by CD3 engagement. Among MAPKs, ERK pathway was essential for homotypic aggregation together with intracytoplasmic Ca(2+).

Myeloid differentiation of human cord blood CD34+ cells during ex vivo expansion using thrombopoietin, flt3-ligand and/or granulocyte-colony stimulating factor.

We investigated the phenotypic changes of human umbilical cord blood (CB) CD34+ cells during ex vivo expansion using thrombopoietin (TPO), flt3-ligand (FL), and/or granulocyte-colony stimulating factor (G-CSF). During ex vivo expansion of CD34+ cells isolated from human CB for up to 5 weeks, surface expression of molecules on the cultured cells including CD64 (Fc gammaRI), CD32 (Fc gammaRII), CD16 (Fc gammaRII), CD11b (MAC-1) and CD18 (beta2-integrin) was analysed by flow cytometry along with simultaneous measurement of apoptosis by 7-aminoactinomycin D staining method. CD64, CD32 and/or CD18 expressing cells appeared in the cultures both with and without the addition of G-CSF until the tenth day. However, without G-CSF, CD16+ fractions did not appear and CD11b+ fractions were not maintained. With G-CSF, the CD16+ or CD11b+ fractions appeared only from the second week. These results suggest that G-CSF is necessary for the late stage of myeloid maturation during which CD16 and CD11b are expressed.

Requirement of MEF2D in the induced differentiation of HL60 promyeloid cells.

The regulatory role of MEF2 (myocyte enhancer binding factor 2) proteins in nonmuscle tissues has not been well characterized. We examined the expression of MEF2 family members, namely, MEF2A, -B, -C, and -D, in the differentiation of HL60 promyeloid cells and observed the remarkable increase in the expressions of MEF2A and MEF2D proteins during the differentiation process into monocytes. To examine the role of MEF2, we expressed a dominant-negative form of MEF2D, without its transactivation domain, in HL60 cells. When the HL60 cell line expressing the mutant MEF2D was induced to differentiate by VitD(3) treatment, cell surface expression of CD14 and the ability to reduce NBT, which are important characteristics of differentiated monocytes, were significantly decreased compared with control HL60 cells. These results show that MEF2D is required in the differentiation process along the monocyte/macrophage lineage,

Expression and purification of the crystalline surface layer protein of Rickettsia typhi.

The crystalline surface layer (S-layer) protein (SLP) of Rickettsia typhi is known as the protective antigen against murine typhus. We previously reported a cloning and sequence analysis of the SLP gene of R. typhi (slpT) and showed that the open reading frame of this gene encodes both the SLP and a 32-kDa protein. To express only the SLP from this gene, the putative signal sequence and the 32-kDa protein portion were removed from the slpT. This protein was expressed in Escherichia coli as a fusion protein, consisting of the SLP and maltose binding protein. The recombinant protein reacted strongly with polyclonal antiserum of a patient with murine typhus.

Cloning and sequence analysis of the gene encoding the crystalline surface layer protein of Rickettsia typhi.

The nucleotide sequence of the gene (slpT) encoding the crystalline surface layer protein (SLP) of Rickettsia typhi was determined. The slpT gene consists of 4935 bp coding for a 1645-amino-acid (aa) protein containing a predicted signal peptide at the N terminus. The size of the predicted SLP exceeds the observed size (135 kDa) on SDS-PAGE. The N-terminal aa sequence of the 32-kDa protein of R. typhi reported by Hackstadt et al. [Infect. Immun. 60 (1992) 159-165] was found in the C-terminal portion of the deduced aa sequence, suggesting that the product of slpT is processed into the mature SLP and the 32-kDa protein.

Partial vs. total footplate removal in stapedectomy: a comparative study.

A comparative study of the postoperative stapedectomy results for 264 ears with partial footplate removal (PFR) and for 106 ears with total footplate removal (TFR) was performed with reference to decibel gain in three specific frequency ranges, air-bone gap closure, speech threshold and speech discrimination and incidence of postoperative complications. The data confirm a small but consistently greater decibel gain for PFR cases in the 2000-8000 Hz range; the decibel gain in the 250-1000 Hz range is virtually identical for PFR and TFR cases. Air-bone gap closure and speech results also indicate a somewhat better average result in PFR as compared TFR cases. The permanency of speech discrimination results is examined.

Primary malleus fixation: diagnosis and treatment.

Primary malleus fixation occurs in an otherwise normal middle ear without evidence of congenital deformity and without chronic inflammatory changes. It occurs in the latter decades of life and is frequently associated with sensorineural presbycusis. We believe it is a ligament ankylosis with osteoarthritis related to the aging process. The diagnosis of malleus fixation is facilitated through the use of a modified Siegle pneumatic otoscope in conjunction with the Zeiss binocular microscope. The literature pertaining to this subject as well as the more historical reports are reviewed. Goodhill has written extensively on malleus fixation. The audiologic test results in the fixed malleus cases reviewed for this study often presented a misleading picture, sometimes mimicking stapedial otosclerosis with a characteristic Carhart's notch and sometimes indistinguishable from sensorineural presbycusis. Usually speech discrimination scores fell in the very good to excellent range. Weber tests, whether performed by tuning forks or audiometrically, almost always lateralized to the suspect ear. Impedance frequently failed to conform to the expected fixed malleus pattern of low static compliance and absent acoustic reflexes; there was an equal number of low compliance and normal range compliance tympanograms and 15% of the total number of our cases had abnormally high compliance tympanograms. Stapedial reflexes are normally expected to be absent with lateral ossicular fixation, but this was not a consistent finding with contralateral test stimulation. The decision for surgical treatment is dependent on the audiological findings and the potential hearing gain. The technique described consists of the removal of the incus and the head of the malleus and the reconstruction of a sound conducting pathway from the handle of the malleus to the mobile stapes or from the mobile stapes to the under surface of the tympanic membrane using a prosthesis-ossicle arrangement. Malleus fixation occurs far more often than it is diagnosed. Surgical correction can result in a worthwhile hearing gain even when the air-bone gap is narrow or nonexistent. The technique of ossicular reconstruction is dictated by the anatomical findings. Some form of autograft ossicular reconstruction from the malleus handle to the stapes is most frequently utilized. Otosclerosis with stapes fixation sometimes causes a lateral ossicular fixation due to degenerative disease and fibrosis. In this instance a stapedectomy is performed as the primary procedure with subsequent revision as necessary to eliminate the lateral obstruction.