Use of a human plaque-forming cell assay to study peripheral blood bursa-equivalent cell activation and excessive suppressor cell activity in humoral immunodeficiency.

A plaque assay that detects human mononuclear blood cells producing immunoglobulin (Ig)M antibody to sheep erythrocytes was investigated for its usefulness in studying B-cell activation and regulation in 24 patients with humoral immunodeficiency. Cells from 3 of 15 patients with common variable agammaglobulinemia produced some plaques (range 40--160/10(6) cells; normal range 80--1240/10(6)), but those from the other 12, from all 7 with x-linked agammaglobulinemia and from the 2 with x-linked immunodeficiency with hyper-IgM failed to produce any detectable plaques. In co-cultures of patient and normal cells a very good correlation was seen between results of the plaque assay and an IgM biosynthesis assay in detecting excessive suppressor cell activity. Cells from 7 of 15 common variable agammaglobulinemics, from 3 of 7 x-linked agammaglobulinemics, and from both patients with hyper-IgM caused significant suppression of IgM biosynthesis and(or) plaque formation by normal cells. The observations in the last two groups and discordance for excess suppressor activity in identical twins with common variable agammaglobulinemia suggest that the activity develops secondarily to whatever their primary defects may be. Culturing non-T cells from common variable agammaglobulinemics exhibiting excessive suppressor cell activity with normal T cells resulted in plaque formation in four of five patients so studied; in all five the suppressor activity was found in the T-cell population. The availability of a plaque assay for the study of blood cells from immunodeficient patients provides a new probe to examine the cellular nature of such defects.

Hyper IgM syndrome associated with defective CD40-mediated B cell activation.

Recent studies show that most patients with X-linked hyper IgM syndrome have defects in the gene for CD40 ligand. We evaluated 17 unrelated males suspected of having X-linked hyper IgM syndrome. Activated T cells from 13 of the 17 patients failed to bind a soluble CD40 construct. In these patients, the sequence of CD40 ligand demonstrated mutations. By contrast, T cells from the remaining four patients exhibited normal binding to the CD40 construct. Sequencing of the cDNA for CD40 ligand from these patients did not show mutations. The possibility that hyper IgM syndrome in these four patients was due to abnormalities in the B cell response to CD40-mediated signals was examined. Peripheral blood lymphocytes were stimulated with anti-CD40 alone, IL4 alone or anti-CD40 plus IL4. In comparison with B cells from controls or patients with hyper IgM syndrome and mutant CD40 ligand, B cells from the patients with hyper IgM syndrome and normal CD40 ligand were defective in their ability to secrete IgE (P < 0.02) or express activation markers, CD25 and CD23 (P < 0.02) in response to stimulation with anti-CD40. The failure of these B cells to respond to CD40-mediated activation could not be attributed to a generalized deficiency in B cell activation because IL4 induced normal up-regulation of CD23 and CD25 expression. These findings indicate that hyper IgM syndrome may result from defects in expression of CD40 ligand by activated T cells or defects in CD40-mediated signal transduction in B cells.

Fatal Epstein-Barr virus infection in a child with acute lymphoblastic leukemia in remission.

A 9-year-old white boy developed a fatal primary Epstein-Barr virus (EBV) infection while receiving chemotherapy for acute lymphoblastic leukemia in remission. Histopathological findings at the height of the proliferative phase of the illness were compatible with a virally induced hemophagocytic syndrome. The infection spontaneously converted to complete aplasia of the bone marrow and lymph nodes. Serological studies disclosed that the patient had no antibodies to EBV prior to the infection, but during the acute phase he showed a spectrum and titers of antibodies to EBV-specific antigens characteristic of a current primary EBV infection. A lymph node biopsy obtained 5 weeks after onset revealed Epstein-Barr nuclear antigen in approximately 50% of the cells. The boy's condition deteriorated rapidly, with disseminated candidiasis resulting in cardiorespiratory failure and death. Lymph nodes obtained at autopsy no longer contained Epstein-Barr nuclear antigen-positive cells.

Diffuse large-cell lymphoma of B-cell origin and deficient T-cell function in a patient with rheumatoid arthritis.

An increased incidence of non-Hodgkin's lymphoma has been described in patients with rheumatoid arthritis. Mechanisms related to abnormal immune regulation have been postulated, but no patients with rheumatoid arthritis and lymphoma have been previously well characterized immunologically. We describe here a patient with long-standing rheumatoid arthritis in whom a B-cell diffuse large-cell lymphoma developed. He was found to have a severe T-cell immunodeficiency and evidence of persistent Epstein-Barr virus infection. Epstein-Barr nuclear antigen was not found to be present within lymphoma cells. The combination of defective T-cell function and persistent Epstein-Barr virus infection may have predisposed this patient with rheumatoid arthritis to the development of a malignant clone of B lymphocytes.

Alterations in immunologic measurements in patients experiencing early hepatic allograft rejection.

We compared lymphocyte subsets, the T4:T8 ratio, and assays of lymphocyte function in 11 recipients of hepatic allografts who showed signs of early graft rejection (less than 3 weeks after transplant) with 13 patients who had no evidence of graft rejection. Both patient groups had similar values for lymphocyte subsets, lymphocyte transformation in the presence of phytohemagglutinin, and in vitro immunoglobulin synthesis prior to allografting. Nonrejectors had a decline in both T3 and T4 cells one week after transplant. Those who had evidence of graft rejection did not show a significant decline in either of these T cell subsets. The T4:T8 ratio declined in 11/13 nonrejectors in the week after transplant but in only 4/11 rejectors (P less than 0.05). Monitoring immunologic markers in blood may represent a means of determining which subjects are at greatest risk for developing early hepatic allograft rejection.

Production of a factor, or factors, suppressing IL-2 production and T cell proliferation by Sertoli cell-enriched preparations. A potential role for islet transplantation in an immunologically privileged site.

Isolated islet allografts survive indefinitely in the abdominal testis of nonimmunosuppressed diabetic rats. The predominant feature of these testes is that the presence of Sertoli cells, but not Leydig cells, is required for extended survival of the islet allografts. Sertoli cells cultures were therefore established in vitro and we examined the effects of the conditioned media on Con A--stimulated spleen lymphocyte proliferation. These studies revealed that a product(s) secreted by Sertoli cells inhibits Con A-stimulated lymphocyte proliferation in a dose-dependent manner. The synthesis of this product is both temperature-dependent, occurring predominantly at 37 degrees C, and hormone-dependent, requiring the presence of follicle stimulating hormone, in the culture medium. We further examined the mechanism of inhibition of lymphocyte proliferation and showed that Sertoli cell-enriched media inhibit the production of IL-2 in a dose-dependent manner. Furthermore, the finding that the addition of exogenous IL-2 is not able to reverse this inhibition indicates that the Sertoli cell-enriched media inhibit both IL-2 production and IL-2 responsiveness of T lymphocytes.

Immunization status of hospitalized preschool children: risk factors associated with inadequate immunization.

OBJECTIVES: The purposes of this study were to determine the accuracy of the immunization histories of hospitalized preschool children, assess the sociodemographic factors associated with delayed immunizations, and interview parents or guardians concerning their views on ways of improving immunization delivery. METHODS: The immunization status of 215 preschool children admitted to a pediatric hospital was determined by interviewing parents or guardians regarding their children's immunization histories. The patient's immunization records were subsequently reviewed for confirmation. The admitting physician's history also was reviewed to determine whether the patient's immunization status had been noted. Finally, parents or guardians of all children studied were interviewed to assess their views on ways of improving the delivery of immunization services. RESULTS: Only 44% of the 215 preschoolers evaluated were adequately immunized. Among those between 2 and 5 years of age, 52% were fully immunized. Only 17% of those who were inadequately immunized could have been completely updated if given an immunization at discharge. The admitting physician failed to document the immunization status of 22% of the patients. Thirty percent of the parents gave inaccurate information concerning the immunization status of their children. Most parents felt that the provision of transport (30%) or formal remainders (21%) would enhance immunization rates. Multiple regression analysis showed that a history of missed opportunity to immunize, male gender, lack of transportation, and lack of day care attendance were significant predictors of delayed immunization. CONCLUSIONS: Resident physicians should be more stringent in documenting the immunization status of all admitted preschoolers so that those found to be delayed could be updated before discharge. A hospital policy of updating underimmunized children at discharge and reporting the immunization status of all discharged patients to their primary care provider could help improve the immunization coverage in this population. Transportation for routine health maintenance and telephone or mailed remainders might further improve the immunization status of inner-city children.

Intravenous methylprednisolone efficacy in status asthmaticus of childhood.

Forty-nine nonsteroid-dependent children hospitalized with status asthmaticus were randomized to receive IV placebo or methylprednisolone treatment (1 mg/kg every six hours). All patients received nebulized isoetharine inhalations and continuous IV aminophylline infusion. Twenty-four hours after admission, the methylprednisolone-treated patients demonstrated a greater rate of improvement in their clinical scoring index than did placebo-treated children. However, the duration of hospital stay was not significantly shortened. Twenty-eight of the patients performed serial bedside spirometry at 0, 12, 24, and 36 hours after admission. The methyl-prednisolone-treated patients experienced a more rapid recovery from peripheral airway obstruction as measured by forced expiratory flow rate during 25% to 75% of forced vital capacity (FEF25-75). The magnitude and rate of improvement in FEF25-75 was significantly greater at 36 hours (P less than .05) and independent of changes in peak expiratory flow rate, forced vital capacity, or forced expiratory volume in the first second of forced vital capacity. Placebo-treated patients had a higher incidence of asthma relapse within 4 weeks of discharge (eight v two relapses, P less than .05). Findings of this study indicate that IV corticosteroid therapy is beneficial in treating pediatric status asthmaticus.

Adjacent-2 disjunction of a maternal t(9;22) leading to duplication 9pter----q22 and deficiency of 22pter----q11.2.

The proposita presented at birth with multiple congenital anomalies including craniofacial anomalies, bilateral cleft lip and palate, abnormalities of the urogenital system, talipes equinovarus, and the DiGeorge sequence. Cytogenetic investigation showed a 46,XX,-22,+der(9)t(9;22)(q22;q11.2) karyotype. The mother, maternal uncle, and maternal grandmother of the infant are carriers of a reciprocal balanced translocation involving chromosomes 9 and 22 at regions q22 and q11.2, respectively. The unbalanced karyotype seen in the proposita arose due to an adjacent-2 disjunction of the quadrivalent in the mother. Prenatal diagnosis of the second pregnancy of this woman showed a similar karyotype. Review of the literature shows that adjacent-2 disjunction may occur preferentially when certain chromosomes are involved in translocations.

Functional aspects of T-lymphocyte subsets in pregnancy.

Several recent reports have suggested that a decrease in circulating T helper cells may contribute to the relative immunodeficiency of pregnancy. To investigate the significance of these findings, 90 pregnant women were evaluated. The results of this study indicate that although pregnant women have a decreased proportion of T helper cells, they do have adequate T helper cell function as determined by an in vitro immunoglobulin synthesis assay and a T-lymphocyte colony-forming assay. Based on these studies it is unlikely that decreased numbers of T helper cells are primarily responsible for the immunodeficiency of pregnancy.

Effects of N-trifluoroacetyladriamycin-14-valerate (AD-32) on human bladder tumor cell lines.

We have compared the in vitro activity of N-trifluoroacetyladriamycin-14-valerate (AD-32) and doxorubicin hydrochloride (ADR) on the clonal growth of human bladder tumor cell lines (HBTCL). In order to determine the relatively toxicity of ADR and AD-32 on hematopoietic stem cells, CFU-GM assays were set up using 10 normal human bone marrow samples. The mean lethal dose for 50% of the colonies (LD-50) for ADR was 1.6 +/- 1.4 microM and that for AD-32, 3.9 +/- 4.9 microM (P less than 0.55), suggesting that these agents have similar bone marrow toxicity. Both drugs produced enhanced inhibition of clonal growth of HBTCL with increasing C X Ts. The spectrum of activity of the two drugs was similar against a panel of seven HBTCL. The activity of ADR was inhibited at 4 degrees C while the activity of AD-32 was unaffected by temperature. ADR was more effective against HBTCL in the log growth phase than the plateau phase while the reverse was found using AD-32. Verapamil was found to enhance the activity of both ADR and AD-32 against a HBTCL (T24), found to be resistant to both agents. The lipophilic properties of AD-32, along with its enhanced activity when used over prolonged periods of time and its activity against tumor cells in the plateau phase, suggest that AD-32 could be useful in the management of patients with superficial bladder cancer.

Formation of pseudoislets from human pancreatic cultures.

We have successfully developed a technique for culturing human islet cells obtained from the cadaveric pancreata of children. Within 24-48 h of in vitro culture, collagenase-digested human pancreatic tissue formed epithelioid monolayers. Scattered within these monolayers were insulin-positive cells, as detected by immunocytochemical and dithizone staining. Treatment of the beta cell-containing epithelioid-cell monolayers with EDTA resulted in the formation of spherical cellular clusters, i.e., pseudoislets. These pseudoislets differed from isolated islets of Langerhans in that they showed a more peripheral distribution of insulin-positive cells. Our studies have demonstrated that insulin-positive cells can be detected in monolayers obtained from human pancreata 3-4 weeks after culture. When exposed to varying concentrations of glucose, these cells secreted insulin. The development of this in vitro technique for culturing human pancreatic islet tissue could provide a model for systematically studying in vitro islet function.

Hereditary angioedema: association with IgA deficiency and otolaryngologic disorders.

Hereditary angioedema (HAE) is an autosomal dominant disorder characterized by recurrent edema of the oropharynx, the extremities, and by abdominal pain. This disorder is caused by a defect in the C1 esterase inhibitor (C1 INH) which inhibits the first component of complement (C1). Four generations of a family are described and laboratory data of five individuals of this family are given as these individuals presented for general otolaryngologic procedures. The mortality of acute laryngeal edema is described to vary from 6% to 54% and may necessitate a tracheotomy as a life saving measure A deficiency of IgA is also noted in this family, and to our knowledge, this is the first time this has been shown in association with HAE. Present therapy consists of long utilization of danazol, an attenuated androgen. Recently a partially purified C1 INH has been reported for acute episodes of HAE, and preliminary results are promising.

E-rosette formation, total T-cells, and lymphocyte transformation in infants receiving intravenous safflower oil emulsion.

There has been much concern about impaired immune function in children receiving infused lipid emulsions. Immunologic studies were carried out on 15 infants maintained on parenteral nutrition with intravenous safflower oil emulsion as part of the infusate. Significant increases in percentage rosette formation, total circulating T-cells, and mitogenesis to phytohemagglutinin and pokeweed mitogen were demonstrated after only 1 wk of lipid infusion. Additional parenteral nutrition did not further increase any immunologic parameter. These results suggest that infused safflower oil emulsion does not adversely alter cellular immune function.

Lymphocyte subsets in children with aplastic anemia.

Twenty-four patients with aplastic anemia (AA) were evaluated with immunologic studies. Patients with posthepatitis AA had decreased proportions of T4 cells, increased proportions of T8 cells, and decreased T4:T8 ratios. Patients with constitutional AA had varied immunologic findings. There were no significant abnormalities in lymphocyte subsets in patients with idiopathic AA. Of the 12 patients who were treated with antithymocyte globulin (ATG), six (50%) responded. Nine of these patients are living 3-27 months after treatment; three of the nine are transfusion dependent. T-cell subsets were not helpful in predicting the response to ATG. Measurements of other immunologic mediators may be more useful with regard to pathophysiology and prognosis in AA.

Evaluation of immune status in pediatric recipients of hepatic orthografts.

Survival in children following hepatic transplantation is better than in adults. We evaluated the immunologic status of both pediatric and adult patients prior to and following transplantation, to determine if there were differences in immunologic status that might contribute to this phenomenon. Studies included determination of mononuclear cell subsets, including T cells, T helper cells, T suppressor cells, and monocytes. In addition, we evaluated immune function by studying in vitro immunoglobulin (Ig) synthesis. Following transplantation, the T8 (cytotoxic/suppressor) cells in pediatric patients did not decline, whereas in adults they did (P less than .05). In vitro Ig synthesis in both adult and pediatric patients declined following transplantation. By 4 weeks, the adult group had begun to recover, whereas in the pediatric group there was still significant suppression (P less than .05). These studies suggest that there is decreased immune function in children who are recipients of hepatic allografts. Such findings could contribute to better outcome of hepatic allografting in children.

Histologic appearance of the islet organoid.

The authors have attempted to develop a hybrid organoid capable of supporting the engraftment of human pancreatic islet tissue transplants. In this report, they we report the histologic appearance of this organoid structure for implantation periods up to 16 days. Both pancreatic duct-like and glandular cells were present inside the organoid. Insulin positive cells were identified within the pancreatic tissue. Although polymorphonuclear (PMN) cells were seen in association with tissue necrosis, the authors observed no lymphocyte infiltration in the area of the xenografted human pancreatic islet tissue in the implanted organoid. Endothelial cell growth factor induced neovascularization inside the organoid sufficient to support the engraftment of transplanted pancreatic islet tissue. This organoid model may provide an alternative for clinical pancreatic transplantation.