Failure to transfer multiple sclerosis into severe combined immunodeficiency mice by mononuclear cells from CSF of patients

To confirm the reported transfer of multiple sclerosis (MS) by CSF cells, we injected CSF cells from six MS patients in the exacerbation stage into the cisterna magna of 18 severe combined immunodeficiency mice. No clinical neurologic abnormalities or light- or electron-microscopic pathologic changes were present in any transferred mice, and the reported results could not be reproduced.     

Arrested rearrangement of TCR V beta genes in thymocytes from children with X-linked severe combined immunodeficiency disease

Human X-linked severe combined immunodeficiency disease (SCID) is an immunodeficiency disorder in which T cell development is arrested in the thymic cortex. B lymphocytes in children with X-linked SCID seem to differentiate normally. X-linked SCID is associated with a mutation in the gene that encodes the IL-2R gamma-chain. Because TCR-beta gene recombination is a pivotal initial event in T lymphocyte ontogeny within the thymus, we hypothesized that a failure to express normal IL-2R gamma could lead to impaired TCR-beta gene recombination in early thymic development. PCR was used to determine the status of TCR-beta gene-segment rearrangements in thymic DNA that had been obtained from children with X-linked SCID. The initial step in TCR-beta gene rearrangement, that of D beta to J beta recombination, was readily detected in all thymus samples from children with X-linked SCID; in contrast, V beta to DJ beta gene rearrangements were undetectable in the same samples. Both D beta to J beta and V beta to DJ beta TCR genes were rearranged in the thymic tissues obtained from immunologically normal children. We conclude that TCR beta-chain gene rearrangement is arrested in children with X-linked SCID. Our results suggest a causative relationship between the failure of TCR beta-chain gene rearrangements to proceed beyond DJ beta rearrangements and the production of a nonfunctional IL-2R gamma-chain.     

Marked osteoblastopenia and reduced bone formation in a model of multiple myeloma bone disease in severe combined immunodeficiency mice

Increased generation of reactive oxygen species (ROS) and low levels of antioxidants may cause morbidity in premature infants on supplemental oxygen. Glutathione (GSH)-dependent antioxidant systems protect against ROS, and regenerating GSH from GSH disulfide (GSSG) by the flavoenzyme GSH reductase (GR) is essential for the optimal function of this system. Previously, we have observed enhanced resistance to t-butyl hydroperoxide (t-BuOOH) in Chinese hamster ovary cells stably transfected with a vector (leader sequence GR [LGR]) for human GR cDNA that contained a functional synthetic mitochondrial targeting signal. The present studies were designed to investigate adenovirus-mediated gene transfer of LGR to H441 cells and resistance of such cells to t-BuOOH. Adenovirus-mediated transfection of H441 cells with LGR increased total GR activities more than 11-fold (mitochondria more than 10-fold and cytosolic more than 7-fold) and protected against t-BuOOH cytotoxicity, as indicated by lower fractional release of cellular lactate dehydrogenase (LDH) than was observed in wild-type untransfected cells (CON) or in cells transfected with a control gene (human manganese superoxide dismutase in the antisense orientation [DOS]) (*LGR 6.6 +/- 1.7; DOS 16 +/- 1.8; CON 16.6 +/- 0.7% LDH release). In addition, cells transfected with LGR retained higher GSH/GSSG ratios (*LGR 66 +/- 0.4; DOS 47 +/- 1; CON 52.6 +/- 2.3) and released less GSH + GSSG to the media in response to challenge with t-BuOOH (*LGR 0.05 +/- 0.01; DOS 0.08 +/- 0.01; CON 0.07 +/- 0.01 nmol/mg of protein) than did wild-type cells or cells transfected with a control vector, indicating an enhanced ability of the LGR cells to reduce GSSG formed in response to exposure to t-BuOOH. In conclusion, adenovirus-mediated gene transfer of LGR enhanced cellular GR activities and protected H441 cells from oxidant stresses.     

Expansion of Philadelphia chromosome-negative CD3(+)CD56(+) cytotoxic cells from chronic myeloid leukemia patients: in vitro and in vivo efficacy in severe combined immunodeficiency disease mice

We have developed culture conditions for the efficient expansion of cytotoxic effector cells from peripheral blood mononuclear cells (PBMNCs) by the timed addition of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and the monoclonal antibody (MoAb) OKT3. These cells, termed cytokine-induced killer (CIK) cells, are composed primarily of T cells, and the population of cells with the greatest cytotoxic activity is an otherwise rare population of CD3(+)CD56(+) cells that expand dramatically under these culture conditions. CIK cells were expanded from PBMNCs from 13 patients with chronic myeloid leukemia (CML). These cultures contained a variable number of T cells at the start of the culture (median 44%, range 1% to 64%), yet after 21 to 28 days of culture, virtually all of the cells were CD3(+) T cells (median 97%, range 90% to 99%). The CD3(+)CD56(+) subset of cells expanded significantly (median 25-fold, range 2.2- to 525-fold). CIK cells from all patients showed cytotoxicity against the tumor cell lines OCI-LY8 and K562. In four patients the expanded CIK cells suppressed colony growth of autologous CML blast cells and myeloid progenitor cells. Allogeneic CIK cells from normal donors also suppressed CML colony growth but did not inhibit growth of normal hematopoietic colonies. Twelve of the 13 cultures were exclusively composed of Philadelphia (Ph)-negative cells and one culture had 1 out of 20 Ph-positive metaphases after 4 weeks in culture. Intracellular cytokine production was assayed by fluorescence-activated cell sorter (FACS), and the expanded T-cell cultures produced IL-2, IFN-gamma, and tumor necrosis factor-alpha (TNF-alpha), but not IL-4. Both the CD4(+) and CD8(+) subsets secreted this cytokine profile. To test the in vivo activity of the expanded CIK cells, CML was engrafted into severe combined immunodeficiency disease (SCID) mice using matrigel. After 4 weeks, 4 x 10(7) autologous CIK cells were injected intravenously by tail vein injection into groups of mice, and the animals were sacrificed after a total of 18 weeks. Bcr-abl was detected in the bone marrow or spleen of 5 out of 6 control mice and only 2 out of 13 mice who received the autologous CIK cells (P = .02). In an additional series of animals, the mice did not engraft with CML but instead developed large human Epstein-Barr virus-associated lymphomas by 12 weeks. The mice who received autologous CIK cells at 4 weeks had either no tumor (5) or small tumors (5), whereas all 10 mice that received CIK cells at week 8 developed lymphomas; however, these were not as large as in the 10 control mice who did not receive CIK cells (P = . 03). This study shows that CIK cells, which are Ph chromosome-negative, can be expanded from patients with CML and have potent in vitro and in vivo efficacy against autologous tumor cells.     

Differential Hsp70 plasma-membrane expression on primary human tumors and metastases in mice with severe combined immunodeficiency

Fibrates are used to lower plasma triglycerides and cholesterol levels in hyperlipidemic patients. In addition, fibrates have been found to alter the plasma concentrations of fibrinogen, plasminogen activator inhibitor-1 (PAI-1) and apolipoprotein A-I (apo A-I). We have investigated the in vitro effects of fibrates on fibrinogen, PAI-1 and apo A-I synthesis and the underlying regulatory mechanisms in primary monkey hepatocytes. We show that fibrates time- and dose-dependently increase fibrinogen and apo A-I expression and decrease PAI-1 expression in cultured cynomolgus monkey hepatocytes, the effects demonstrating different potency for different fibrates. After three consecutive periods of 24 h the most effective fibrate. ciprofibrate (at 1 mmol/l), increased fibrinogen and apo A-I synthesis to 356% and 322% of control levels, respectively. Maximum inhibition of PAI-1 synthesis was about 50% of control levels and was reached by 1 mmol/l gemfibrozil or ciprofibrate after 48 h. A ligand for the retinoid-X-receptor (RXR), 9-cis retinoic acid, and specific activators of the peroxisome proliferator-activated receptor-alpha (PPARalpha), Wy14,643 and ETYA, influenced fibrinogen, PAI-1 and apo A-I expression in a similar fashion, suggesting a role for the PPARalpha/RXRalpha heterodimer in the regulation of these genes. When comparing the effects of the various compounds on PPARalpha transactivation activity as determined in a PPARalpha-sensitive reporter gene system and the ability of the compounds to affect fibrinogen, PAI-1 and apo A-I antigen production, a good correlation (r=0.80; p <0.01) between PPARalpha transactivation and fibrinogen expression was found. Apo A-I expression correlated only weakly with PPARalpha transactivation activity (r=0.47; p=0.24), whereas such a correlation was absent for PAI-1 (r=0.03; p=0.95). These results strongly suggest an involvement of PPARalpha in the regulation of fibrinogen gene expression.     

Generation of intestinal mucosal lymphocytes in SCID mice reconstituted with mature, thymus-derived T cells

Transfer of peripheral lymph node lymphocytes to SCID mice leads to the long term establishment of mucosal T lymphocytes within the epithelium and lamina propria of the small and large intestines. Analysis of engrafted intraepithelial lymphocytes (IEL) showed that they had acquired a surface phenotype that in several respects is typical of IEL. In addition, the functional profile of engrafted IEL derived from lymph node T cells was similar to that of normal IEL; as the donor-derived T cells exhibited a strong cytolytic activity, a poor proliferative response to mitogenic stimuli, and a tendency to home and expand specifically in the intestine upon transfer to secondary SCID recipients. Optimal engraftment of intestinal T cells required bacterial flora, as the number of lymphocytes was greatly reduced in SCID recipients with a reduced flora. These results demonstrate that mature, thymus-derived T cells can migrate to the intestine and become functionally specialized to the intestinal milieu. The acquisition of phenotypic markers characteristic of the intestinal microenvironment by engrafted cells suggests that T cell migration of lymphocytes to the SCID intestine is not aberrant, but it may reflect processes that are ongoing in immunocompetent mice. Furthermore, these data suggest that the homing and/or expansion of typical, thymus-derived T cells in the intestine may be driven by luminal Ags such as those derived from bacterial flora.     

Deficiency of adenosine deaminase not associated with severe combined immunodeficiency

The 12-year-old Kung ("Bushman') boy from South West Africa who has marked deficiency of red cell adenosine deaminase has been found to have 2 to 3% of enzyme activity in red blood cells, 10 to 12% in leukocytes, and 10 to 30% in cultured fibroblasts. The enzyme has ADA 1 electrophoretic mobility: SV40 transformation of cultured fibroblasts caused a decrease of "tissue ADA' and an increase in "red cell ADA' isozymes. A battery of investigations revealed that the child has normal humoral and cellular immunity. A family study showed that a sibling had the same level of red cell ADA and the parents had intermediate levels. Studies of the Kung population from which the child comes have shown that the allele responsible for the condition, and which we designate ADA8, is polymorphic.     

Long-term malignant hematopoiesis in human acute leukemia bone marrow biopsies implanted in severe combined immunodeficiency mice

Bone marrow (BM) trephine biopsies from 15 pediatric patients with acute lymphoid (ALL) or myeloid (AML) leukemia were engrafted subcutaneously into severe combined immunodeficiency (SCID) mice conditioned by 200 cGy total-body irradiation. Implants were harvested 5 to 19 weeks later for histologic, cytologic, and/or flow cytometric analysis of the residing marrow. Eighteen of 19 grafts contained viable human leukemic cells to various extents as assessed by one or more of these methods. Thirteen of 14 implants analyzed by flow cytometry included high numbers of tumor cells, accounting for 85% to 100% of the total nucleated cells in seven of them. Histologically, engrafted marrow samples exhibited areas of blastic infiltration, and tumor-specific gene rearrangements were retrieved in long-term engrafted biopsies. Importantly, engrafted mice remained perfectly healthy even 5 months posttransplantation, and no human tumor cell dissemination was detected in the hematolymphoid and nonhematopoietic tissues at the time of autopsy. These results demonstrate that human malignant hematopoiesis can be sustained long-term in its original, intact marrow stromal environment transplanted in appropriately conditioned immunodeficient mice.     

Growth of human T-cell lineage acute leukemia in severe combined immunodeficiency (SCID) mice and non-obese diabetic SCID mice

Primary leukemic cells from patients with acute lymphoblastic leukemia (ALL) can be injected intravenously into mice with severe combined immunodeficiency (SCID) to create a model of human leukemia. Leukemic cells disseminate to murine tissues in a clinicopathologic pattern similar to that seen in humans. Thus far, reports of engraftment of lymphoid leukemia in SCID mice have mainly been from patients with B-cell lineage ALL, for which engraftment occurs more frequently with cells from high-risk patients. There are few data on the engraftment of T-cell lineage ALL in SCID mice. Leukemic cells from 19 patients (16 adult and three pediatric) with T-cell lineage ALL were injected into SCID mice, with overt engraftment of 12 cases (63%). Engraftment of leukemia in SCID mice was associated with earlier death due to leukemia of the patient donors (P < .01, log-rank test). The recently developed non-obese diabetic (NOD)/SCID mouse may expand the uses of the SCID model. Cells from the seven patients with T-cell lineage ALL that failed to cause leukemia in SCID mice were injected into NOD/SCID mice. Overt leukemia engraftment was observed in all seven cases. Thus, growth of human T-cell lineage ALL cells in SCID mice was associated with a high-risk patient group. However, this association was not observed when NOD/SCID mice were used, suggesting that this model would no longer predict patients likely to die early of leukemia, but may provide a more realistic system for studying the biology and treatment of the disease.     

Development of autologous, oligoclonal, poorly functioning T lymphocytes in a patient with autosomal recessive severe combined immunodeficiency caused by defects of the Jak3 tyrosine kinase

Defects of the common gamma chain subunit of the cytokine receptors (gamma c) or of Jak3, a tyrosine kinase required for gamma c signal transduction, result in T-B+ severe combined immunodeficiency (SCID). However, atypical cases, characterized by progressive development of T lymphocytes, have been also reported. We describe a child with SCID caused by Jak3 gene defects, which strongly but not completely affect Jak3 protein expression and function, who developed a substantial number (> 3,000/microL) of autologous CD3+CD4+ T cells. These cells showed a primed/activated phenotype (CD45R0+ Fas+ HLA-DR+ CD62L(lo)), defective secretion of T-helper 1 and T-helper 2 cytokines, reduced proliferation to mitogens, and a high in vitro susceptibility to spontaneous (caused by downregulation of bcl-2 expression) as well as activation-induced cell death. A restricted T-cell receptor repertoire was observed, with oligoclonal expansion within each of the dominant segments. These features resemble those observed in gamma c-/y and in Jak3-/- mice, in which a population of activated, anergic T cells (predominantly CD4+) also develops with age. These results suggest that residual Jak3 expression and function or other Jak3-independent signals may also permit the generation of CD4+ T cells that undergo in vivo clonal expansion in humans; however, these mechanisms do not allow development of CD8+ T cells, nor do they fully restore the functional properties of CD4+ T lymphocytes.     

Cavitary pulmonary lesions in patients infected with human immunodeficiency virus

The differential diagnosis of cavitary pulmonary lesions in individuals infected with human immunodeficiency virus (HIV) is broad, especially in patients with advanced disease. In patients with Pneumocystis carinii pneumonia, cavitation is an uncommon manifestation of a common disease. It is unusual in patients with pulmonary cryptococcosis, coccidioidomycosis, and histoplasmosis but occurs frequently in patients with invasive pulmonary aspergillosis. In patients with pulmonary tuberculosis, cavities are more common during earlier stages of HIV disease, when cellular immunity is relatively preserved. Mycobacterium avium complex is an uncommon cause of lung disease and infrequently produces cavities. However, Mycobacterium kansasii, is often associated with cavitation. Cavities can complicate any bacterial pneumonia and are especially common with pneumonia due to Pseudomonas aeruginosa, Nocardia asteroides, and Rhodococcus equi. Noninfectious causes of cavitary lesions are rare, but cavitary lesions caused by pulmonary Kaposi's sarcoma and non-Hodgkin's lymphoma have been reported. Because of the broad differential diagnosis and because most cavities are caused by treatable opportunistic infections, a definitive diagnosis is essential.     

Rhesus thymic/liver xenografts in severe combined immunodeficient mice: immunologic reconstitution and intrathymic infection with simian immunodeficiency virus

By serving as host recipients of xenografts from both humans and animals, severe combined immunodeficient (SCID) mice have become valuable to many laboratories interested in examining the pathophysiology of different diseases. To gain insight into the usefulness of the SCID mutation in retrovirus research, rhesus monkey fetal hematolymphoid tissues (liver and thymus) were used to construct a SCID-rhesus chimeric mouse (SCID-rh) and were engrafted in the renal capsule. The size and maturation of the thymic engrafts were monitored grossly, histologically, and immunologically. SCID mice were tolerant to rhesus tissues, and thymic engrafts contained thymocytes at different stages of maturation and differentiation that had morphologic features similar to age-matched rhesus thymus. Mature single positive CD2+, CD4+, and CD8+ T lymphocytes that were phenotypically similar to rhesus T lymphocytes were present at low levels (2% to 5%) in the peripheral blood and at moderately higher levels (7% to 15%) in the spleens of SCID-rh mice obtained between 12 and 15 weeks after thymus/liver engraftment. Within 3 weeks after engraftment, > 85% of the thymocytes in the thymic engrafts were immature double positive CD4+CD8+ T cells. The highest number of positive cells were seen in thymic engrafts obtained at 12 to 18 weeks. During these weeks, > 90% of the cells were double positive (CD2+CD4+, CD2+CD8+, and CD4+CD8+). After infection of the engrafted thymus tissue with simian immonodeficiency virus (SIVmac239), PCR analysis revealed successful viral infection of engrafts at 2 and 4 weeks after infection. No significant histopathologic and flow cytometric changes were observed in the thymic engrafts at 2 and 4 weeks after infection. An unrelated lesion of thymic lymphomas involving the SCID host thymus was seen in 12% of the mice. The data presented herein suggest that the SCID-rh is a valuable model for specific studies related to thymus-retrovirus interaction and that it could be used for further studies. The results are discussed in relation to current knowledge of thymus involvement during simian and human immunodeficiency virus infection.     

Impaired human responses to tetanus toxoid in vitamin A-deficient SCID mice reconstituted with human peripheral blood lymphocytes

Vitamin A deficiency is associated with increased childhood morbidity and mortality from respiratory and diarrheal diseases. In order to evaluate the effect of vitamin A on human antibody responses, we developed a vitamin A-deficient severe combined immunodeficient (SCID) mouse model. Vitamin A-deficient mice were produced by depriving them of vitamin A at day 7 of gestation. Mice were reconstituted with human peripheral blood lymphocytes (huPBL) from tetanus toxoid immune donors at 6 weeks of age and immunized with tetanus toxoid at 6 and 8 weeks of age. Secondary human antibody responses were determined 10 days later. The geometric mean human anti-tetanus toxoid immunoglobulin G concentrations were 3.75 micrograms/ml for the deficient mice and 148 micrograms/ml for controls (P = 0.0005). Vitamin A-deficient mice had only a 2.9-fold increase in human anti-tetanus toxoid antibody compared with a 74-fold increase in controls (P < 0.01). Supplementation with vitamin A prior to reconstitution restored human antibody responses to normal. These data suggest that vitamin A deficiency impairs human antibody responses. We speculate that impaired responses could increase susceptibility to certain infections. Furthermore, we propose that effects of other nutritional deficiencies on the human immune system could be evaluated in the SCID-huPBL model.     

Transgenic mice expressing human immunodeficiency virus type 1 in immune cells develop a severe AIDS-like disease

Panic disorder is a common and disabling condition which frequently leads to excessive reliance upon medical facilities. It is also closely associated with the development of agoraphobia. Medical approaches implicate disturbances of ascending brain noradrenergic and serotonergic systems, and support related pharmacotherapies. Contemporary psychological approaches focus upon misinterpretations of bodily sensations and an undue appreciation of the risk of life-threatening illness, and support cognitive/behavioral psychotherapies. A synthesis is possible by developing the view that the implicated ascending aminergic systems normally play a part in "effortful" or context-sensitive behavior. A relative failure of this under conditions of heightened arousal might be responsible for the rigid patterns of fear, belief, and behavior that characterize these patients. Clinical and research implications are discussed.     

Three novel mutations in the interleukin-2 receptor gamma chain gene in four Japanese patients with X-linked severe combined immunodeficiency

Three novel mutations in the IL-2R gamma chain gene were identified in four Japanese patients with X-linked severe combined immunodeficiency by direct sequence analysis of polymerase chain reaction (PCR) amplified DNA fragments.     

The pathophysiological significance of nondesmoglein targets of pemphigus autoimmunity. Development of antibodies against keratinocyte cholinergic receptors in patients with pemphigus vulgaris and pemphigus foliaceus

OBJECTIVES: To determine whether nondesmoglein (non-Dsg) autoantibodies are pathogenic and whether they recognize keratinocyte cholinergic receptors that control cell adhesion because antikeratinocyte autoimmunity in patients with pemphigus vulgaris is not limited to the development of autoantibodies to Dsg. DESIGN: To determine whether non-DSg autoantibodies are pathogenic, we sought to induce pemphigus in genetically engineered neonatal mice lacking Dsg 3 using pemphigus vulgaris IgGs that did not cross-react with Dsg 1. To determine whether pemphigus autoimmunity involves keratinocyte cholinergic receptors, the latter were separated from cell membranes of human keratinocytes, tagged with the covalent label [3H]propylbenzilylcholine mustard, and used as an antigen in a radioimmunoprecipitation assay of 34 pemphigus vulgaris and 6 pemphigus foliaceus serum samples. SETTING: The dermatologic clinics of the University of Minnesota, Minneapolis; the Mayo Clinic, Rochester, Minn; and the University of California-Davis Medical Center, Sacramento. PATIENTS: Serum samples were collected from 34 patients with pemphigus vulgaris and 6 patients with pemphigus foliaceus (aged 31-89 years) and from 7 age-similar patients of both sexes with nonpemphigus blistering or the following immune-mediated conditions: pemphigoid gestation, bullous drug eruption, lupus erythematosus, erythema nodosum, urticaria, acute contact dermatitis, and skin ulcers. MAIN OUTCOME MEASURES: Clinical, laboratory, and histopathologic findings. RESULTS: Extensive skin blistering accompanied by the Nikolsky sign and suprabasilar acantholysis was induced in the Dsg3null mice that received pemphigus, but not normal human IgGs. In the radioimmunoprecipitation assays for reactivity with cholinergic receptors, the mean radioactivity precipitated by pemphigus serum samples significantly exceeded both normal- and disease-control levels (P = .001-.02). The mean individual levels of radioactivity precipitated by 34 pemphigus vulgaris and pemphigus foliaceus serum samples (85%) exceeded control values by a mean of approximately 2.6 times. CONCLUSIONS: Autoantibodies to keratinocyte cell-surface molecules other than Dsg 1 and Dsg 3 can induce clinical features of pemphigus vulgaris. Patients with pemphigus vulgaris and those with pemphigus foliaceus develop IgG antibodies that precipitate radiolabeled cholinergic receptors. Because these receptors control keratinocyte adhesion and motility, their inactivation by autoantibodies may elicit intracellular signals that cause disassembly of desmosomes, leading to acantholysis and blistering.