Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Sequence Count Data”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 433 records · Page 24Linked to original sources
PubMed1997

Rhizobium gallicum sp. nov. and Rhizobium giardinii sp. nov., from Phaseolus vulgaris nodules.

Thirty-one strains of two new genomic species (genomic species 1 and 2) of rhizobia isolated from root nodules of Phaseolus vulgaris and originating from various locations in France were compared with reference strains of rhizobia by performing a numerical analysis of 64 phenotypic features. Each genomic species formed a distinct phenon and was separated from the other rhizobial species. A comparison of the complete 16S rRNA gene sequences of a representative of genomic species 1 (strain R602spT) and a representative of genomic species 2 (strain H152T) with the sequences of other rhizobia and related bacteria revealed that each genomic species formed a lineage independent of the lineages formed by the previously recognized species of rhizobia. Genomic species 1 clustered with the species that include the bean-nodulating rhizobia, Rhizobium leguminosarum, Rhizobium etli, and Rhizobium tropici, and branched with unclassified rhizobial strain OK50, which was isolated from root nodules of Pterocarpus klemmei in Japan. Genomic species 2 was distantly related to all other Rhizobium species and related taxa, and the most closely related organisms were Rhizobium galegae and several Agrobacterium species. On the basis of the results of phenotypic and phylogenetic analyses and genotypic data previously published and reviewed in this paper, two new species of the genus Rhizobium, Rhizobium gallicum and Rhizobium giardinii, are proposed for genomic species 1 and 2, respectively. Each species could be divided in two subgroups on the basis of symbiotic characteristics, as shown by phenotypic (host range and nitrogen fixation effectiveness) and genotypic data. For each species, one subgroup had the same symbiotic characteristics as R. leguminosarum biovar phaseoli and R. etli biovar phaseoli. The other subgroup had a species-specific symbiotic phenotype and genotype. Therefore, we propose that each species should be subdivided into two biovars, as follows: R. gallicum biovar gallicum and R. gallicum biovar phaseoli; and R. giardinii biovar giardinii and R. giardinii biovar phaseoli.

Colony Count, Microbial↗
PubMed1995

Horse gammaglobulin-induced thrombocytopenia in anaphylaxis involving sequestration and activation of platelets.

Thrombocytopenia as well as hemoconcentration and leukopenia followed by leukocytosis were induced after HoGG challenge on HoGG-sensitized mice. Thrombocytopenia was induced within 2 min and sustained for 1 day. HoGG-induced thrombocytopenia was not observed until day 10 after sensitization; mice challenged with HoGG dose > or = 10 micrograms developed thrombocytopenia. Two types of thrombocytopenia were observed in appropriately sensitized mice. HoGG induced thrombocytopenia at 2 min and 60 min, whereas, alpha-macroglobulin induced thrombocytopenia at 2 min, the platelet count of which returned to normal within 60 min. Poly (Glu60Ala30Tyr10) did not induce thrombocytopenia at 2 min or 60 min. The tracing study by 3H-serotonin labelled platelets demonstrated the 2 min-sequestration of platelets in lungs or livers. The HoGG-induced sequestration of platelets at 2 min was blocked by high dose heparin or Cobra Venom factor. Platelet activation at 60 min was partially inhibited by dexamethasone, rhodostomin synthetic peptide 45-59, or platelet activation factor antagonist (WEB 2086). Furthermore, the thrombocytopenia could be transferred by heat (56 degrees C, 4h) treated immune sera. This suggests that HoGG-induced, non-IgE-mediated thrombocytopenia in anaphylaxis involves sequestration and activation of platelets. The sequestion in lungs occurs within 2 min and can be inhibited by high dose heparin or Cobra Venom factor. The activation of platelets involves platelet activation factor, and fibrinogen receptor.

Amino Acid Sequence↗
PubMed1999

Symbiosis of Aeromonas veronii biovar sobria and Hirudo medicinalis, the medicinal leech: a novel model for digestive tract associations.

Hirudo medicinalis, the medicinal leech, is applied postoperatively in modern medicine. Infections by Aeromonas occur in up to 20% of patients unless a preemptive antibiotic treatment is administered. The associated infections demonstrate the need for a better understanding of the digestive tract flora of H. medicinalis. Early studies reported the presence of a single bacterial species in the digestive tract and suggested that these bacteria were endosymbionts contributing to the digestion of blood. In this study, we cultivated bacteria from the digestive tract and characterized them biochemically. The biochemical test results identified the isolates as Aeromonas veronii biovar sobria. This species identification was supported by sequence comparison of a variable region of the genes coding for 16S rRNA. In a colonization assay, a rifampin-resistant derivative of a symbiotic isolate was fed in a blood meal to H. medicinalis. The strain colonized the digestive tract rapidly and reached a concentration similar to that of the native bacterial flora. For the first 12 h, the in vivo doubling time was 1.2 h at 23 degreesC. After 12 h, at a density of 5 x 10(7) CFU/ml, the increase in viable counts ceased, suggesting a dramatic reduction in the bacterial growth rate. Two human fecal isolates, identified as Aeromonas hydrophila and A. veronii biovar sobria, were also able to colonize the digestive tract. These data demonstrate that the main culturable bacterium in the crop of H. medicinalis is A. veronii biovar sobria and that the medicinal leech can be used as a model for digestive tract association of Aeromonas species.

Aeromonas↗
PubMed2001

Identification and characterization of a second superoxide dismutase gene (sodM) from Staphylococcus aureus.

A gene encoding superoxide dismutase (SOD), sodM, from S. aureus was cloned and characterized. The deduced amino acid sequence specifies a 187-amino-acid protein with 75% identity to the S. aureus SodA protein. Amino acid sequence comparisons with known SODs and relative insensitivity to hydrogen peroxide and potassium cyanide indicate that SodM most likely uses manganese (Mn) as a cofactor. The sodM gene expressed from a plasmid rescued an Escherichia coli double mutant (sodA sodB) under conditions that are otherwise lethal. SOD activity gels of S. aureus RN6390 whole-cell lysates revealed three closely migrating bands of activity. The two upper bands were absent in a sodM mutant, while the two lower bands were absent in a sodA mutant. Thus, the middle band of activity most likely represents a SodM-SodA hybrid protein. All three bands of activity increased as highly aerated cultures entered the late exponential phase of growth, SodM more so than SodA. Viability of the sodA and sodM sodA mutants but not the sodM mutant was drastically reduced under oxidative stress conditions generated by methyl viologen (MV) added during the early exponential phase of growth. However, only the viability of the sodM sodA mutant was reduced when MV was added during the late exponential and stationary phases of growth. These data indicate that while SodA may be the major SOD activity in S. aureus throughout all stages of growth, SodM, under oxidative stress, becomes a major source of activity during the late exponential and stationary phases of growth such that viability and growth of an S. aureus sodA mutant are maintained.

Amino Acid Sequence↗
PubMed2000

Proviral HIV-1 dynamics and evolution in patients receiving efficient long-term antiretroviral combination therapy.

OBJECTIVES: Different experimental approaches have shown that, despite plasma viral loads under the threshold of detection, HIV-1 frequently continues to replicate in patients receiving potent antiretroviral therapy. However, whether this low-grade viral replication is sufficient for the generation of new major quasispecies has not been studied. Thus, in order to evaluate the extent of variation in the major proviral HIV-1 population, we monitored proviral DNA sequences in such patients over a time period of up to 30 months. METHODS: DNA was extracted from peripheral blood mononuclear cells (PBMC) and the V3 region was amplified by nested polymerase chain reaction (PCR) and directly sequenced. Additionally, both HIV-1 RNA and DNA levels and CD4+ T-lymphocyte counts were monitored. RESULTS: Analysing the V3 gene sequences of 17 patients, we observed a sequence evolution in nine patients. Interestingly, the majority of these changes (77%) occurred in the first interval following the initiation of therapy and despite signs of ongoing replication the proviral DNA levels continued to decrease in all patients. CONCLUSIONS: Our data suggest that, although available data report that HIV-1 continues to replicate in patients with undetectable viraemia, the extent of viral replication in many of these patients is not sufficient to result in changes in the major viral population.

Adult↗
PubMed1998

Extrathymic T cell differentiation in the human intestine early in life.

It is clear from experimental studies in mice that T cell maturation can occur outside the thymus, especially in the intestine. There is little sound evidence so far that extrathymic T cell maturation occurs to any significant extent in human gut, and, postnatally, there is abundant evidence that the gut mucosa is an immune effector organ. Here, we describe a large population of T lymphocytes in human fetal intestinal mucosa that are proliferating (Ki67+) in the absence of foreign Ag (CD3+, Ki67+ lamina propria lymphocytes (LPL) 22 +/- 1.8% and CD3+, Ki67+ intraepithelial lymphocytes (IEL) 9.1 +/- 1.4%), that express the T cell activation markers CD103, HLA-DR, and L-selectin(low), and that express mRNA transcripts for pre-TCR-alpha. There is also a substantial proportion of CD7+ LPLs that do not express CD3 (CD3-7+, 14 +/- 7% of all LPLs) in the fetal gut that may be differentiating into CD3+ cells. Rearranged TCR-beta transcripts of fetal LPLs, IELs, and paired blood lymphocytes were cloned and sequenced, and virtually no overlap of clonality was observed between blood and intestine, suggesting that gut T cells may not be derived from the blood. In addition, 30 days after engraftment of SCID mice with fetal intestine, CD3-7+ cells, proliferating T cells, and pre-TCR-alpha transcripts were abundant, and there is a threefold increase in CD3+ IELs. These data show that in the human intestine before birth a population of precursor T cells exists that may be differentiating into mature T cells in situ.

Aging↗
PubMed1997

Expression of mucin-associated tumor antigens is altered by cell density.

Mucin-associated sialylated Lewis antigens are implicated in tumor cell metastasis and are used in several tests for pancreatic cancer. Despite their clinical importance, little is known about the structures of the oligosaccharides of pancreatic cancer mucins or about the regulation of their synthesis or of the synthesis of their protein cores. In this study, we examined the effects of culture at high cell density on the expression of these antigens in the SW1990 human pancreatic cancer cell line. Mucins from cells that were 2.5 weeks post-confluent had increased expression of sialyl-Lewis(a) and Lewis(x) antigens but reduced expression of the DU-PAN-2 antigen (NeuAc alpha2,3Galbeta1,3GlcNAc-Gal-R) when compared to mucins from 1 day post-confluent cells. Sialyl-Lewis antigens differ from the DU-PAN-2 antigen by the presence of an additional fucose. Mucins from 2.5-week cells also had increased binding to lectins specific for fucose, such as AAL and UEAI, with no apparent change in the binding of lectins specific for sialic acids. Metabolically radiolabeled O-linked oligosaccharides with sialyl-Lewis(a) antigenic reactivity eluted from Bio-Gel P-10 in the region of sialylated and sulfated oligosaccharides. Oligosaccharides eluted from QAE-Sephadex (2 mM Tris base) in a pattern suggesting the presence of 1, 2 and 3 or more negative charges per oligosaccharide. Even after desialylation and desulfation, oligosaccharides eluted from Bio-Gel P-10 with apparent molecular sizes greater than glucose oligomers of 12 units. Culture of SW 1990 cells at high density also increased the steady-state levels of mRNA for mucins MUC1, 2, 4, 5 and 6. In summary, after prolonged culture at high cell density, SW1990 cells have qualitative changes in their oligosaccharides that may be due to up-regulation of fucosyltransferases.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed1996

Phenotypic and functional analysis of B lymphopoiesis in interleukin-7-transgenic mice: expansion of pro/pre-B cell number and persistence of B lymphocyte development in lymph nodes and spleen.

Transgenic mice in which mouse interleukin (IL)-7 cDNA is expressed under the control of the mouse major histocompatibility complex (MHC) class II (E alpha) promoter develop a lymphoproliferative disease characterized by the early polyclonal expansion of T cells followed in many cases by the development of lymphomas of immature B cells. Here, we have analyzed B cell development in these transgenic mice. Phenotypic analysis using monoclonal antibodies to B220, IgM, IgD, c-kit, IL-7 receptor, MHC class II, AA4.1, CD19, CD23, CD25, CD40 and CD43 shows that B lymphopoiesis in the bone marrow is dramatically altered and the number of pro/pre-B and immature B cells is significantly increased. Interestingly, pro/pre-B and immature B cells persist in the spleens of adult transgenic mice and are also present in lymph nodes and blood. Cell cycle analysis of lymph node cells shows that subpopulations of developing B cells retain the cell cycle profiles of their bone marrow counterparts. Limiting dilution analysis shows that the number of clonable pre-B cells is significantly increased and that at limiting dilution, growth of transgenic pre-B cells is still dependent on exogenous IL-7. Using semiquantitative polymerase chain reaction (PCR) and in situ hybridization, the level of IL-7 transcripts in the spleen was found to decrease between 2 and 4 weeks in control mice with levels in transgenics mice being approximately 50 times greater. These transgenic mice represent an interesting model with which to study the effects of IL-7 overexpression in the bone marrow and raise interesting questions regarding the regulation of B lymphopoiesis in normal mice.

Animals↗
PubMed2004

Circulation and homing of melanoma-reactive T cells to both cutaneous and visceral metastases after vaccination with monocyte-derived dendritic cells.

Anticancer immune therapies aim at the induction of tumor-specific T cells, which ultimately should kill tumor cells. The effector cells should, therefore, not only exert cytotoxic activity but also home to and infiltrate the tumor site. Hence, monitoring of immune modulating therapies should not be restricted to the circulating pool of peripheral blood mononuclear cells (PBMC) but also include tumor-infiltrating lymphocytes (TIL), as well as the correlation of these findings to the clinical course. We report here on the longitudinal immunologic workup of a melanoma patient who developed remarkably potent ex vivo detectable antimelanoma cytotoxic T-cell (CTL) responses after vaccinations with autologous peptide-pulsed dendritic cells. Such potent CTL responses to multiple tumor antigens have, to the best of our knowledge, not been described previously in melanoma patients, neither spontaneously nor after any therapy. This patient first experienced a transient response to therapy but finally succumbed to disease progression and died. Progression was associated with the decline of the numbers of tumor-reactive T cells in circulation and at skin metastases in addition to the loss of MHC class I antigens. The immunologic analysis revealed that fully functional tumor-specific T cells were present in the peripheral blood of this patient during the phase of a relatively stable disease, and in situ tetramer staining demonstrated that these cells were also accumulated at cutaneous and visceral tumor sites. Furthermore, comparative clonotype mapping of PBMC and TIL depicted an overlapping TCR repertoire usage among these 2 compartments. Since strong CTL responses as observed in this patient are the goal of cancer vaccination but are so far only rarely observed, the thorough analysis of patients exhibiting either exceptional clinical and/or immunologic responses appears critical to understanding how vaccine therapies work and can be further improved.

Aged↗
PubMedDate not supplied

Immunobiology of a synthetic luteinizing hormone receptor peptide 21-41.

Immunization of adult male rabbits with a synthetic luteinizing hormone-receptor peptide (LH-RP; representing amino-acids 21-41 of the extracellular domain of the rat LH receptor) resulted in production of high-titer antibodies capable of interacting with particulate and cell-based LH receptors. The antibody produced was able to inhibit binding of 125I-labeled human chorionic gonadotropin (hCG) to a particulate sheep luteal LH receptor preparation by 40%-50%. Maximal inhibitory activity was correlated with high antibody titer. Immunocytometry revealed that the antibody could directly bind to cells having LH receptors, such as rat granulosa and Leydig cells. The antibodies recognized a 77-kilodalton membrane protein in Western blots of mouse testicular extracts. Interaction of endogenous Leydig cell LH receptor with the LH-RP antibody resulted in both hormone agonist and antagonistic activities. The hormone-mimicking activity (increase in serum testosterone over control) was confined only to the early phase of immunization when the antibody titer was low. Blockade of LH receptor during the later part of immunization resulted in a significant reduction in serum testosterone over controls and inhibition of spermatogenesis. DNA flow cytometry showed that a specific and significant inhibition of meiosis (transformation of primary spermatocytes to round and elongated spermatids P < .01) and spermiogenesis (transformation of round spermatids to elongated spermatids P < .0001) occurred following blockade of LH function.

Amino Acid Sequence↗
PubMed1995

Scintillation proximity assay to measure binding of soluble fibronectin to antibody-captured alpha 5 beta 1 integrin.

A scintillation proximity assay (SPA) has been developed to measure binding of alpha 5 beta 1 integrin, a heterodimeric cell-surface adhesion receptor, to fibronectin. This assay utilizes an anti-beta 1 integrin monoclonal antibody to simultaneously capture alpha 5 beta 1 from a cellular lysate and couple the integrin to anti-mouse IgG antibody-coated SPA beads for detection of 125I-fibronectin binding. The assay does not require prior purification of alpha 5 beta 1 nor physical separation of bound and free 125I-fibronectin. Chinese hamster ovary cells that stably overexpress human alpha 5 integrin (CHO#7 cells) were used as a source of alpha 5 beta 1 fibronectin receptor. Using the anti-hamster beta 1 monoclonal antibody 7E2 to capture alpha 5 beta 1 from a CHO#7 cell lysate, this SPA assay allowed measurement of specific 125I-fibronectin binding as defined by displacement by the Arg-Gly-Asp containing peptide GRGDSP or the anti-human alpha 5 antibody P1D6. IC50 values for displacement of 125I-fibronectin binding by GRGDSP and the novel cyclic peptides cRGDGF, cRGEGF, and cRRETAWA were 2.6, 0.045, 3.2, and 37 microM, respectively. Specific 125I-fibronectin binding to alpha 5 beta 1 from C8161 human melanoma cells was also measured using anti-human beta 1 antibodies. This method should be generally useful to measure cell-free ligand binding to receptors that are difficult to purify.

Amino Acid Sequence↗
PubMed1999

Quantification of epitope-specific MHC class-II-restricted T cells following lymphocytic choriomeningitis virus infection.

CD4(+) T cells are critical for the control of many viruses; however, the numbers of virus-specific CD4(+) cells that are expanded following infection are unknown. We have addressed this issue by enumerating virus-specific, MHC class-II-restricted T cells following infection of mice with lymphocytic choriomeningitis virus (LCMV). We have found that the numbers of T cells that produce interferon-gamma in response to stimulation with three different class-II-restricted LCMV epitopes increase from undetectable numbers in noninfected animals to between 4 x 10(5) and 2 x 10(6) cells per spleen at the peak of the T cell response. This contrasts with the numbers of virus-specific class-I-restricted T cells which expand to 1 x 10(7) to 2 x 10(7) cells per spleen during the same time period. We could not reproducibly detect virus-specific class-I-restricted or class-II-restricted T cells that produced interleukin-4 at any time following LCMV infection, indicating that infection with this virus induces a predominantly type 1 cytokine response. In contrast to the rapid decrease in the numbers of class-I-restricted T cells, the numbers of LCMV-specific class-II-restricted T cells declined gradually following the peak of the T cell response. We demonstrate, therefore, that following infection with LCMV there is expansion of both class-I-restricted and class-II-restricted virus-specific T cells; however, the degree of expansion of class-II-restricted T cells is substantially less than that observed for class-I-restricted cells. Furthermore, the downregulation phase of the class-II-restricted response is protracted compared with the precipitous contraction of the antiviral CD8(+) T cell response.

Amino Acid Sequence↗
PubMed1992

Comparative effects of a recombinant and a mutein type of granulocyte colony stimulating factor on the growth of Meth-A fibrosarcoma with 5-fluorouracil chemotherapy.

The present study was designed to evaluate the effects of a recombinant human G-CSF (rhG-CSF) and a mutein G-CSF (KW-2228) on leucopenia and tumor growth in mice treated with 5-fluorouracil (5-FU). In normal mice, the number of leucocytes (white blood cell, WBC) reached the peak 12 hours after a single injection of either type of G-CSF and decreased to the normal level after 24 hours. Daily administration induced a continuous increase in the WBC count, however, administrations at intervals did not. Meth-A fibrosarcoma was subcutaneously inoculated into the backs of syngeneic BALB/c mice. The mice were treated with 5-FU alone or with G-CSFs. Chemotherapy with 5-FU alone resulted in leucopenia and an insignificant inhibition of tumor growth. The conjunctive administration of G-CSFs with 5-FU resulted in a significantly augmented inhibition of tumour growth, and leukopenia was not seen. This augmenting effect was more prominent with KW-2228. These results suggest that in 5-FU chemotherapy G-CSFs may be beneficial in restoring the number of leucocytes from leucopenic state and in augmenting the tumor inhibitory effect. Furthermore, KW-2228 may be more beneficial than the natural type rhG-CSF.

Amino Acid Sequence↗
PubMed2005

Transient expression and insertional mutagenesis of Puccinia triticina using biolistics.

The fungal genus Puccinia contains more than 4,000 species. Puccinia triticina, causal agent of wheat leaf rust, is an economically significant, biotrophic basidiomycete. Little is known about the molecular biology of this group, and tools for understanding gene function have not yet been established. A set of parameters was established for the transient transformation of urediniospores. The expression of three heterologous promoters (actin, elongation factor 1-alpha, and Hss1, Heat Shock 70 protein), derived from Puccinia graminis, was evaluated along with the potential for insertional mutagenesis. The UidA (GUS) gene was used as a marker for transient expression. When transferred into P. triticina urediniospores, transient expression was observed across four helium pressures using one size of gold and three sizes of tungsten microprojectiles. Each of the three promoters displayed strong transient expression in germinated urediniospores; however, higher numbers of GUS-positive urediniospores were observed when either the actin or Hss1 promoters were used. Possible concomitant insertional mutagenesis of several avirulence genes was selected in wheat cultivars harboring the cognate resistance genes. Using a linearized cloning plasmid, stable integration into the genome was achieved as demonstrated by PCR and sequencing analysis.

Base Sequence↗
PubMed1993

Dexamethasone treatment fails to reduce oxygen-induced lung injury in the preterm guinea pig. Effects on pulmonary inflammation and antioxidant status.

Dexamethasone (10 mg/kg/day) or vehicle was administered in a randomized, controlled fashion to 3-day preterm guinea pigs exposed to either 21% oxygen or 95% oxygen for 72 hr and maintained in room air for a further 96 hr. Treatment with dexamethasone had no effect on survival of preterm pups maintained in either 21% or 95% O2. Dexamethasone treatment reduced the growth rate of pups, the effect occurring earlier (0-3 days) in 21% O2-treated pups than in 95% O2-treated pups (5-7 days). Exposure to 95% O2 reduced the survival rate of preterm animals (73% vs 100%, P < 0.05). Surviving pups developed acute lung injury, characterized by the accumulation of a protein-rich exudate in the alveoli and an infiltration of inflammatory cells, particularly neutrophils into the lung. Dexamethasone treatment attenuated the pulmonary inflammatory cell infiltration, in particular neutrophils, both during oxygen exposure (16.4 x 10(4) vs 9.4 x 10(4)/mL; P < 0.05) and following return to ambient conditions (28.0 x 10(4) vs 5.1 x 10(4)/mL; P < 0.05). Elastase activity in bronchoalveolar lavage fluid, which was primarily of neutrophil origin, was unchanged by dexamethasone treatment. Dexamethasone-treated pups had increased pulmonary antioxidant enzyme activities (Cu/Zn-superoxide dismutase; Mn-superoxide dismutase, catalase and glutathione peroxidase) during recovery from oxidative injury. Although there was both a marked reduction in numbers of neutrophils in the lung and elevated pulmonary antioxidant enzyme activities in dexamethasone-treated pups, the degree of microvascular permeability, as determined by both the lung wet weight/dry weight ratio and the presence of plasma proteins in the lavage fluid, was unchanged. Combined, these results imply that dexamethasone, although capable of blunting the influx of neutrophils to the hyperoxia-exposed lung and inducing antioxidant defences in the immature lung, cannot modify the progression of acute oxygen-induced injury of the immature lung.

Amino Acid Sequence↗
PubMed1995

Quantification of antigen specific CD8+ T cells using an ELISPOT assay.

An ELISPOT assay to detect and determine the number of antigen specific CD8+ T cells was standardized using cloned murine CD8+ T cells specific for the epitope SYVPSAEQI of a rodent malaria antigen. This assay is based on the detection of IFN-gamma secretion by single cells after their stimulation with antigen. The interferon secretion is visualized as spots revealed by using enzyme labeled anti-IFN-gamma monoclonal antibodies. Using known numbers of cloned murine CD8+ T cells it was determined that the assay detects 80-95% of these CD8+ T cells. The optimal culture conditions for the stimulation of the CD8+ T cells were determined and the antigen concentration, number of antigen presenting cells and supplement of growth factors required to perform the assay were defined. This ELISPOT assay can be performed with spleen cells from immunized mice, and provide the precise number of antigen specific CD8+ T cells present in mixed lymphocyte populations. This method is more sensitive than the chromium-51 release assay, and much simpler than the conventional precursor frequency analysis, providing the number of antigen specific CD8+ T cells in 36-48 h.

Amino Acid Sequence↗
PubMed1996

Murine bone marrow-derived macrophages constitute feeder cells for human B cell hybridomas.

Murine bone marrow-derived macrophages (BMDM), a homogeneous cell population easily obtainable in large quantities and at reproducible quality by in vitro differentiation, were used as feeder cells for human B cell hybridomas after fusion or during recloning. We used as antigens for the in vitro immunization of human B lymphocytes from peripheral blood as well as from tonsils: (i) synthetic peptides representing immunogenic sequences of gp160 and Nef of HIV-1, coupled to the lipopeptide carrier N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2 RS)-propyl]-(R)-cysteinyl(-seryl-seryl) (P3 CSS-[gp160(303-329)] and P3C-nef24), (ii) the toxins saxitoxin and microcystin, coupled to BSA (BSA-STX and BSA-MCYST). After fusion with the mouse-human heteromyeloma CB-F7, we could demonstrate that BMDM exert a strong growth supporting effect on post-fusion cultures, resulting in 81.6% versus 23.6% growth-positive wells for P3C-nef24, and 100% versus 71.2% growth-positive wells for BSA-STX stimulated cells in cultures with and without BMDM, respectively. Furthermore, clones in wells with BMDM grew much more rapidly, resulting in 24.3% versus 3.6%, 98.1% versus 42.2% and 56.7% versus 6.7% of cultures ready for screening 2 weeks after fusion of P3C-nef24, P3CSS-[gp160(303-329)], and BSA-STX stimulated lymphocytes, respectively. Apart from their effect on cell growth, murine BMDM also increased the percentage of immunoglobulin (Ig)-producing cultures after fusion, as shown for BSA-STX stimulated lymphocytes (47.8% versus 6.7%), as well as the percentage of cultures producing specific antibodies, as demonstrated with BSA-MCYST activated cells (42% versus 10%). Finally, recloning efficiencies of two human B cell hybridomas (E 10 and F 2) were raised profoundly by BMDM, resulting in 100% versus 64.2% and 90.9% versus 44.2% growth-positive wells after recloning on a ten cells/well level. As murine BMDM can also be stored in liquid nitrogen without loss of activity, they constitute ideal feeder cells for the establishment of human B cell hybridomas.

Amino Acid Sequence↗
PubMed1993

Didanosine (ddI) and zidovudine (ZDV) susceptibilities of human immunodeficiency virus (HIV) isolates from long-term recipients of ddI.

Thirty HIV isolates, obtained from 15 patients before and after receiving single drug therapy with didanosine (ddI), were examined for sensitivity to ddI and zidovudine (ZDV) using a peripheral blood mononuclear leukocyte (PBML)-based assay. Fourteen of the patients had ARC, one had AIDS and 12 had received previous therapy with ZDV. After a median of 1 year of ddI therapy, isolates were significantly less sensitive to ddI than were isolates obtained prior to therapy (P = 0.03). A decrease in ddI sensitivity was observed in ten of the 15 isolate pairs. In contrast to ddI susceptibilities, sensitivity to ZDV increased over the same period of time (P = 0.03). Additional isolates were obtained from four patients who received ddI monotherapy for 2 years. Three of these isolates demonstrated no change in ddI sensitivity compared to baseline. No correlation could be made in this study between development of decreased ddI sensitivity and serum p24 levels, CD4 counts, or clinical outcome. Decreased ddI sensitivity occurs frequently among HIV isolates obtained from long-term recipients of ddI. This decreased sensitivity is modest in degree and is of unknown clinical significance.

AIDS-Related Complex↗