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Biomedical subjects

A P Knutsen

Publications and source records attributed to A P Knutsen.

At least 19 recordsLinked to original sources
PubMed1998

Expression of macrophage markers by a population of T cells obtained from synovial fluid of a subgroup of patients with juvenile rheumatoid arthritis.

OBJECTIVE: To characterize distinctive lymphoid cell populations in the synovial fluid (SF) of patients with juvenile rheumatoid arthritis (JRA) that have the specific ability to display monocytic markers when cultured in vitro. METHODS: Mononuclear cells obtained from SF of patients with JRA and depleted of adherent macrophages were cultured in vitro in RPMI 1640 medium supplemented with only fetal calf serum (FCS). Phenotypic evaluation of these cells was by flow cytometry and immunohistochemical analysis was by specific fluorochrome labeled antibodies. RESULTS: T cells from a JRA subgroup displayed some typical macrophage attributes, i.e., abundant cytoplasm, adherence to plastic, and phagocytosis of latex beads when cultured in vitro. These cells have the ability to survive in culture for several weeks in RPMI 1640 medium containing only 10% FCS. The macrophage-like T cells rosetted with sheep red blood cells and proliferated when stimulated with phytohemagglutinin or anti-CD3, indicating functional T cell responses. CONCLUSION: Our data indicate that a population of T cells obtained from the SF of a subgroup of patients with JRA exhibited characteristics of macrophages, yet retained their CD3 and T cell receptor expression. Whether this promiscuous behavior is caused by malignant transformation of lymphoid precursor cells or is induced by the concerted effect of a myriad of cytokines and growth factors present in the SF remains unknown. The presence of these cells in the SF of 2 patients with JRA with different onset types raises the question of their function and significance in an autoimmune disorder such as JRA.

Adolescent↗
PubMed1997

The association of HLA-DR alleles and T cell activation with allergic bronchopulmonary aspergillosis.

Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease caused by the mold Aspergillus fumigatus. We previously reported that the majority of T cell clones (TCC) isolated from three ABPA patients, and specific for a dominant Ag of A. fumigatus, Asp f 1, were IL-4-producing CD4+ Th2 cells capable of responding to Ag in association with the HLA-DR subtypes DRB1*1501, *1503, and *1601 for HLA-DR2, and DRB1*1101, *1104, and *1202 for HLA-DR5. In the present study we extended the previous findings to determine whether the observed restriction with the HLA-DR2/5 subtypes held importance in a larger patient population. Serotyping revealed that 16 of 18 ABPA patients were either HLA-DR2, HLA-DR5, or both. Compared with a normal control population, the frequencies of HLA-DR2 (50 vs 22.3%) and HLA-DR5 (44.4 vs 19.8%) were significantly increased in these ABPA patients. Genotype analyses of an additional 15 patients identified the same HLA-DR subtypes previously shown functional for Asp f 1 Ag presentation. The relative avidities of Asp f 1 peptides for the purified HLA-DR subtypes, DRB1*1501 (functional) and DRB1*1502 (nonfunctional), were examined to determine whether differential binding to the HLA-DR subtypes explains successful Ag presentation. Similar low binding avidities were detected for both HLA-DR subtypes, indicating that the functionality cannot be simply explained by differences in binding affinities. Thus, the limited number and their role in Ag presentation emphasizes the possibility that the six identified HLA-DR subtypes are important in the pathophysiology of ABPA.

Alleles↗
PubMed1996

T cell differentiation/maturation of CD34+ stem cells from HIV-seropositive hemophiliacs in cultured thymic epithelial fragments.

The clinical manifestations of AIDS are predominantly due to the cellular and humoral immune dysfunction caused by HIV infection, and thymic dysplasia caused by HIV infection probably contributes to the T cell lymphopenia. In the present study, T cell differentiation and/or maturation was assessed when enriched CD34+ stem cells (SCs or SC) purified from bone marrow of HIV-seropositive hemophiliacs were cocultured with allogeneic cultured thymic epithelial fragments (CTEFs). When HIV-seropositive hemophiliacs' enriched CD34+ SC were cocultured with allogeneic CTEFs, acquisition of the T cell phenotypic markers CD7, CD2, CD3, CD4, CD8 and T cell receptor for antigen (TCR) alpha beta was observed from cells harvested from the culture media peaking at approximately 28 days. Origin of the differentiated and matured T cells from the CD34+ SC was confirmed by labeling the SC with 5-(and -6)-(((4-chloromethyl)benzoyl)amino)tetra-methyl-rhodamine (CMTMR), a fluorescent cytoplasmic dye, and detecting fluorescence in the differentiated and matured T cell by flow cytometry. In one experiment, CMTMR labeling was omitted and double positive CD4+CD8+ and triple positive CD3+CD4+CD8+ thymocytes were identified. These studies confirmed that thymocyte differentiation/maturation from SC had occurred. In addition, T cells obtained from the CD34+ SC and CTEF cocultures proliferated to phytohemagglutinin stimulation maximally with stem cell donor antigen-presenting cells (APCs) and also proliferated to pooled B cells in a mixed lymphocyte culture (MLC). Furthermore, the T cells produced were tolerant to thymus donor B cell HLA antigens (p < 0.025); though there was slight MLC reactivity to autologous stem cell donor B cell HLA compared to thymic B cells (p < 0.025). These T cells demonstrated positive self-alloreactivity to stem cell HLA antigens in four of nine persons, though decreased compared to pool B cell alloantigens. Furthermore, in three experiments, responsiveness to stem cell donor B cells subsequently disappeared upon further duration of CD34+ SC-CTEF coculture. These studies suggested that CD34+ SC gave rise to accessory cells populating the thymus that contributed to HLA restriction. To further evaluate this hypothesis, two different donors of CD34+ SC were cultured simultaneously with thymic epithelial fragments and MLC reactivity was then examined toward APC of the stem cell donors. In these experiments, T cells responded to stimulation with HLA antigens of the pool B cells and did not respond to thymus donor B cells. In six of eight experiments, the chimeric SC-CTEF T cells did not respond to stimulation with B cells of either stem cell donor. These studies suggest that HLA restriction and tolerance were induced by cells of the stem cell donor as well as the thymic epithelial cell HLA antigens. In summary, these studies demonstrated that HIV-infected hemophiliac bone marrow-derived nonadherent CD34+ SC were capable of differentiating and/or maturing into T cells when cocultured in a normal allogeneic thymic environment. Furthermore, the T cells derived from derived CD34+ SC were capable of differentiating into T cells when cocultured in a normal allogeneic thymic environment, proliferated maximally with APCs from the stem cell donor and were tolerant of thymic HLA class II antigens, and to a lesser degree to stem cell donor B cell HLA antigens.

Antigen-Presenting Cells↗
PubMed1996

Arrest of in vitro T cell differentiation of normal bone marrow-derived CD34+ stem cells with thymic epithelial fragments from children with AIDS.

A novel approach is presented to assess the ability of thymic tissues obtained from children with end stage AIDS to attract normal bone marrow (BM)-derived CD34+ (lineage negative) stem cells (SCs) and support lymphopoiesis in vitro. Chemokinesis of BM-derived CD34+ SCs was analyzed by time-lapse videomicroscopy to ascertain whether an alteration in SC motility could contribute to abnormal thymopoiesis under conditions of HIV infection. The migration of SCs derived from an HIV+ donor into thymic tissue was not significantly altered compared to normal controls, as were normal SCs migrating toward thymic epithelial cell monolayers derived from an HIV+ patient. Thymic tissue obtained from children with AIDS contained nests of CD34+ SCs identified by immunofluorescence, indicating SC homing to the thymus is apparently supported in HIV infection. The ability of HIV-affected thymic epithelial fragments to support lymphopoiesis was determined by examining the initial thymocyte populations present, compared to thymocytes produced de novo in T cell-depleted thymic fragments, following a single pulse of lineage negative CD34+ CD38- SCs. In comparison to normal controls, thymocytes derived from the HIV-affected thymic epithelial fragment coculture had an increased percentage of triple negative thymocytes (28% of lymphocytes from HIV-affected tissue versus 1.5% in controls, p < 0.01) and a decreased percentage of double and single positive CD4+ thymocytes. However, CD3+CD8+ TCR alpha beta + expression was comparable to control cultured thymic epithelial fragments indicating that HIV-affected thymic epithelia were capable of supporting the development of the CD8+ lineage. In an effort to extend the information obtained to date from the histological examination of HIV-affected thymic tissue, select patient thymic tissues were maintained in culture to evaluate the capacity of undifferentiated thymic epithelial cell guirlandes to differentiate in vitro. A partial regeneration of certain subpopulations of the thymic epithelium defined by TE-4 monoclonal antibody (mAb) and CDR2 mAbs occurred during the in vitro culture. The epithelial and mesenchymal components of thymic tissues were distinguished by immunostaining for keratins (indicative of epithelium) and vimentin (a mesenchymal marker). Further evaluation of the modulation of HIV thymus, with respect to the testing of new therapeutic strategies on SCs, will be possible with this in vitro model.

ADP-ribosyl Cyclase↗
PubMed1996

Development of a method of thymocyte differentiation of bone marrow-enriched CD34+CD38- cells in postnatal allogeneic cultured thymic epithelia to evaluate immunodeficiency disorders.

An in vitro model of CD34+CD38- stem cell (SC) differentiation in postnatal cultured thymic epithelia fragment (CTEF) cocultures is described. Sequential phenotypic analysis of the progeny of the SC-CTEF demonstrated predominantly thymocytes and minor populations of promyelocytes, monocytes and natural killer cells. Triple-positive CD3+CD4+CD8+, double-positive CD4+CD8+, and mature single-positive CD4+ and CD8+ T cells, which were TCR alpha beta+, were identified indicating normal thymocyte maturation. In kinetic studies, mature single-positive CD4+ T cells increased from 29% of total cells at one week to 54% at four weeks of coculture. These findings demonstrate that coculture of bone marrow-derived SC and allogeneic cultured thymic epithelia in vitro results in continuous normal predominantly thymocyte differentiation. The SC-CTEF cocultures were then infected with two different strains of human immunodeficiency virus. CD4+ thymocytes were markedly decreased. However, inhibition of early thymocyte maturation steps was also suggested by the presence of increased triple-negative and CD44+CD25-CD3-thymocytes and decreased CD44+CD25+ thymocytes. This model system of thymocyte maturation will be useful in the evaluation of primary T cell immunodeficiency disorders, gene therapy of SC and pharmacological augmentation of thymic function.

ADP-ribosyl Cyclase↗
PubMed1996

Abnormal in vitro thymocyte differentiation in a patient with severe combined immunodeficiency-Nezelof's syndrome.

An in vitro coculture model system of CD34+ stem cells and allogenic cultured thymic epithelia fragments was used to evaluate thymocyte differentiation in a 9-month-old child of Amish descent with Nezelof syndrome. Though the patient's stem cells differentiate to acquire normal expression of CD2 and CD7, later steps of maturation were abnormal. There was detectable but reduced expression of CD3 and CD4 phenotypes. CD44+ expression, however, was markedly reduced. CD44 is an adhesion molecule, interacting with the matrix ligands hyaluronan and fibronectin, and is expressed early in thymocyte differentiation and subsequently in mature T cells. It is hypothesized that abnormal expression of CD44 in a variant of severe combined immunodeficiency, Nezelof's syndrome, interferes with normal thymocyte and thymic epithelial interaction, which leads to abnormal thymocyte differentiation.

Antigens, CD34↗
PubMed1996

A 12-year longitudinal study of Aspergillus sensitivity in patients with cystic fibrosis.

STUDY OBJECTIVE: The object of the study was to longitudinally follow immune parameters of Aspergillus fumigatus sensitization so as to predict those at risk for developing allergic bronchopulmonary aspergillosis (ABPA). DESIGN: Patients were evaluated for 5 immune parameters (skin test [ST], positive precipitating antibody [PPN], total IgE, IgE anti-A fumigatus antibody [IgE-Af], and IgG anti-A fumigatus antibody [IgG-Af]) at yearly intervals over a 12-year time period. SETTING: Patients were enrolled and evaluated during routine visits to the cystic fibrosis (CF) clinic at Cardinal Glennon Children's Hospital, St. Louis. PATIENTS: One hundred eighteen patients with documented CF participated. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: Six patients were diagnosed as having ABPA. In the non-ABPA patient group, 42% had a positive ST, 42% were PPN positive, 54% had IgE-Af, 61% had IgG-Af, and 10% had an IgE greater than 1,000 IU/mL at some point in time. However, on follow-up, 18% lost skin reactivity, 54% lost-PPN, 53% lost IgE-Af, 45% lost IgG-Af, and IgE greater than 1,000 IU/mL declined more than 72% in 64% of patients. These losses were spontaneous, without systemic corticosteroid intervention. CONCLUSIONS: Spontaneous diminution and loss of immune parameters in non-ABPA CF patients prevented us from defining a profile of sensitivity likely to result in ABPA. This variability highlights the importance of obtaining follow-up studies and including clinical symptoms when considering the diagnosis of ABPA in patients with CF.

Adolescent↗
PubMed1994

Asp f I CD4+ TH2-like T-cell lines in allergic bronchopulmonary aspergillosis.

Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease caused by bronchial colonization with Aspergillus fumigatus (Af) characterized by elevated serum total and Af-specific IgE levels and eosinophilia. In order to examine T-cell reactivity to Af antigens, six T-cell lines were established from the peripheral blood of patients with ABPA to Asp f I, an 18 kd protein purified from Af extracts. The Asp f I-specific T-cell lines, analyzed by flow cytometry, were 100% CD3+ CD4+. Lymphoproliferative responses of the T-cell lines were specific for Asp f I stimulation, 28,999 cpm (stimulation index = 12.2), and showed no response to tetanus toxoid stimulation, 2178 cpm (stimulation index = 1.1) (p < 0.001). Furthermore, Asp f I-stimulated lymphoproliferation was inhibited in two experiments by monoclonal anti-interleukin (IL)-4 antibody in a dose-response fashion, 78% and 84% inhibition at 5% concentration of anti-IL-4. In contrast, anti-IL-2 antibody did not inhibit Asp f I-stimulated proliferation. Asp f I-stimulated T-cell lines synthesized predominantly IL-4 (mean, 21.5 ng/ml) after 48 hours of culture, and nondetectable quantities of interferon-gamma and IL-2. In summary, Asp f I-specific T-cell lines established from patients with ABPA were characterized as being CD4+ TH2-like in their cytokine synthesis pattern, and secreted IL-4 behaved in an autocrine fashion, stimulating proliferation.

Allergens↗
PubMed1994

Serum anti-Aspergillus fumigatus antibodies by immunoblot and ELISA in cystic fibrosis with allergic bronchopulmonary aspergillosis.

Allergic bronchopulmonary aspergillosis (ABPA) occurs with a prevalence of 5% to 15% in patients with cystic fibrosis (CF). Because of the frequent colonization with Aspergillus fumigatus (Af) in CF, the causative agent of ABPA, antibody reactivity to Af proteins is frequently observed, which obscures the diagnosis of ABPA. Patients with CF are also categorized according to the presence of positive skin test responses to Af and/or the presence of positive precipitins. In this study we used ELISA and immunoblot assay to detect IgE and IgG anti-Af antibodies in patients with CF and ABPA (n = 13) compared with other groups of patients with CF: those with positive skin test and positive precipitin results (n = 18), those with positive skin test and negative precipitin results (n = 14), those with negative skin test and positive precipitin results (n = 10), and those with negative skin test and negative precipitin results (n = 35). IgE and IgG anti-Af antibodies were significantly elevated in patients with ABPA as determined by both immunoblot assay (p < 0.01) and ELISA (p < 0.01). However, detection of Af antibodies by ELISA was more sensitive in discriminating patients with CF and ABPA from patients with CF who had positive skin test and positive precipitin results but lacked radiographic and clinical evidence of ABPA. In patients with CF and ABPA the immunoblot assays demonstrated a multitude of IgE, IgG, and IgA antibody responses to Af proteins, which ranged in molecular weight from 14 kd to greater than 106 kd. The level of IgE anti-Af antibody to individual proteins decreased during remissions of ABPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1994

Antiepileptic drug hypersensitivity syndrome: in vitro and clinical observations.

Four patients who manifested symptoms of the antiepileptic drug (AED) hypersensitivity syndrome during therapy with carbamazepine are reported. In 3 patients, the syndrome was exacerbated after conversion of therapy to another antiepileptic drug with an aromatic ring chemical structure. In vitro lymphocyte transformation studies confirmed cross-reactivity to one or more aromatic AEDs in 3 patients. In all patients, discontinuation of aromatic AEDs resulted in resolution of symptoms and in 3 patients who required continued AED therapy, valproic acid was well tolerated.

Adolescent↗
PubMed1994

The role of ethylene oxide allergy in sterile shunt malfunctions.

Failure of an intact ventriculoperitoneal shunt, in the absence of an overt infection, is often due to its occlusion by cellular debris and/or an abdominal pseudocyst. This failure is thought to be caused by an infection by an organism which is difficult to culture or by some poorly defined allergic response to the shunt materials. Little attention has been directed to the treatment that the shunts receive prior to implantation: specifically, their exposure to ethylene oxide as a means of sterilization. We have found ethylene oxide metabolites in the spinal fluid of children with shunt malfunction months after their systems were implanted. Many of these patients had coincident CSF eosinophilia. In addition, two of the children had detectable serum IgE antibody directed against an albumin-ethylene oxide conjugated protein. Both of these children had several shunt malfunctions within a short period, yet neither child could be shown to have a shunt infection despite multiple cultures. We therefore suggest that in some patients proteins altered by ethylene oxide incite an IgE mediated response which may lead to shunt malfunction.

Adolescent↗
PubMed1993

A 13-month-old child with chronic diarrhea, weight loss, and tachypnea.

We present a case of a patient with common variable immune deficiency presenting as the Letterer-Siwe syndrome (disseminated Langerhans cell histiocytosis). To our knowledge, this is the only known patient with this association. The clinical presentation was chronic diarrhea, weight loss, recurrent infections, hepatosplenomegaly, and interstitial pneumonitis. Laboratory evaluation revealed evidence of immunodeficiency, with agammaglobulinemia and diminished number and function of T cells. The diagnosis of Langerhans cell histiocytosis was confirmed by electron microscopic examination of the lung biopsy specimen demonstrating Birbeck granules in the cells of the infiltrate. It is known that patients with the combined immunodeficiency syndrome may present as disseminated Langerhans cell histiocytosis, and the case presented demonstrates that patients with common variable immune deficiency may similarly present. It is advisable that patients newly diagnosed with Langerhans cell histiocytosis be evaluated to screen for immunodeficiency. Conversely, patients presenting with combined immunodeficiency or common variable immune deficiency may display features of disseminated Langerhans cell histiocytosis. These associations must be considered in newly diagnosed immunodeficient patients.

Chronic Disease↗
PubMed1990

T- and B-cell dysregulation of IgE synthesis in cystic fibrosis patients with allergic bronchopulmonary aspergillosis.

Since Aspergillus fumigatus (Af)-specific and polyclonal serum IgE levels are characteristically elevated in patients with allergic bronchopulmonary aspergillosis (ABPA), we evaluated in vitro regulation of IgE synthesis in cystic fibrosis (CF) patients with ABPA. We studied 11 CF patients with ABPA, 37 patients with positive Af prick skin tests and/or IgG precipitating antibodies (ST/PPT+), and 35 patients with no humoral or skin responses to Aspergillus (ST/PPT-). Mean serum IgE concentration was significantly elevated in CF subjects with ABPA compared to ST/PPT+ and ST/PPT- patients, 2866 vs 303 and 61 IU/ml, respectively (P less than 0.01). In vitro studies demonstrated that ABPA patients' B cells spontaneously synthesized significantly increased amounts of IgE compared to ST/PPT positive and negative subjects, 1980 vs 220 and 13 pg/ml, respectively (P less than 0.01). In addition, preformed B-cell-associated IgE was also significantly elevated in ABPA subjects (P less than 0.01), indicating prior in vivo activation. Supernatant cultures of Af-stimulated T cells from ABPA subjects significantly induced allogeneic B-cell IgE synthesis compared to ST/PPT positive and negative CF subjects, 206 vs 13 and 4 pg/ml, respectively (P less than 0.01). Thus T cells stimulated with Aspergillus antigens secrete cytokines that induce B-cell IgE synthesis in ABPA subjects. B-cell IgE hyperactivity is manifested by in vivo and in vitro increased IgE concentrations. Analyses of T-cell regulation and B-cell IgE synthesis distinguish CF subjects with ABPA from Aspergillus sensitive non-ABPA subjects.

Aspergillosis, Allergic Bronchopulmonary↗
PubMed1990

T cell cytotoxicity in cystic fibrosis: relationship to pulmonary status.

T cell cytotoxicity (CTL) to an allogeneic lymphocyte target was evaluated in patients with cystic fibrosis (CF) before and during pulmonary exacerbations (group 1) compared to another group of CF patients who had stable pulmonary disease activity during their two study periods (group 2). CTL activity was significantly decreased in group 1 subjects studied prior to their pulmonary flares and in group 2 CF patients compared to normal controls at effector:target ratios of 12.5:1 and 6.25:1 (p less than 0.05 and p less than 0.05, respectively). Furthermore, in group 1, CTL lysis was significantly decreased during pulmonary flares compared to before flares at the 25:1 and 12.5:1, effector:target, (p less than 0.05 and p less than 0.05, respectively). T suppressor cell activity as measured by effect on in vitro control B cell IgM synthesis was significantly increased during pulmonary flares (p less than 0.05). Diminished CTL may be partially responsible for persistent colonization of Pseudomonas aeruginosa in CF.

Cystic Fibrosis↗
PubMed1990

Serum immunoglobulins E and G anti-Aspergillus fumigatus antibody in patients with cystic fibrosis who have allergic bronchopulmonary aspergillosis.

Patients with cystic fibrosis frequently have pulmonary colonization with Aspergillus fumigatus (Af) and develop anti-Af immunoglobulin E (IgE) and IgG antibodies. The diagnosis of allergic bronchopulmonary aspergillosis in subjects with cystic fibrosis is difficult because of the high incidence of Af colonization, with development of humoral antibody responses. In this study, we sequentially measured serum anti-Af IgE (Af-E) and IgG (Af-G) antibodies by ELISA in subjects with cystic fibrosis. In subjects with cystic fibrosis who have allergic bronchopulmonary aspergillosis, Af-E and Af-G antibodies were significantly increased when compared with other groups of patients with cystic fibrosis who had positive skin tests or precipitins to Af (or both) (p less than 0.01, p less than 0.01, respectively). In addition, increased Af-E and Af-G levels were sometimes seen in other groups, especially subjects with cystic fibrosis who had positive Af skin tests or precipitin tests, two of whom later developed criteria diagnostic of allergic bronchopulmonary aspergillosis. Thus, serum Af-E and Af-G levels were quantitatively increased in subjects with cystic fibrosis who had allergic bronchopulmonary aspergillosis and thus adjunctive data in diagnosis. However, it also suggested that subclinical pulmonary inflammation may also occur.

Adolescent↗