Negative Siblings Research Articles

The 12th International Immunology and Diabetes Workshop: Orlando, Florida

The 12th International Immunology of Diabetes Workshop was held during April 1993 in Orlando, Florida, to review research progress since the 11th Immunology of Diabetes Workshop meeting in Nagasaki, Japan, one and a half years before. The NOD mouse may have as many as 10 susceptibility genes, including its novel IA major histocompatibility complex antigen and a defective interferon-gamma receptor, whereas human IDDM is so far known to be encoded by cis and trans complementation products of certain DQ genes on chromosome 6q, and a gene in the insulin-like growth factor II region on chromosome 11p. A unique protein regulator of the X box promotor of the highly susceptible DQB1*0302 allele has also been found. Islet cell antibody negative siblings of IDDM patients appear to have lower than expected abilities to secrete insulin in response to intravenous glucose. Sera from patients before and/or after developing IDDM immunoprecipitate two native proteins of 64,000- and 38,000-M(r) glutamic acid decarboxylase (GAD65) reacting to conformational epitopes. However, a multitude of other autoantibodies often reacting to denatured proteins through linear epitopes have also been identified. The first workshop for GAD antibody assays was successfully completed; however, the 38,000-M(r) antigen has not yet been identified. Other autoantibodies reactive to gangliosides and to sulfatides continue to be reported. Insulitis has come to be recognized as a sometimes protective event. Protective insulitis predominates in older lesions. It can be induced by as disparate means as tuberculin antigen administration, by interleukin-4 treatments, by transfer of T-cell lines generated in autologous mixed lymphocyte responses, and by immunization to insulin B-chain, whereas oral islet cell antigens, such as insulin, can delay diabetes onset in the NOD mouse.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased insulin response to glucose in islet cell antibody-negative siblings of type 1 diabetic children.

Measurement of beta-cell function is an important marker of progression to diabetes in individuals at risk for the disease. Although the peak incidence for the disease occurs before 17 years of age, normal values for insulin secretion were not available in this age group. We performed a simplified intravenous glucose tolerance test in 167 normal children, and in 98 islet cell antibody (ICA)-negative and 12 ICA-positive siblings of diabetic patients. Their age range was 1-16 yr. The first phase of insulin secretion, evaluated as the sum of plasma insulin concentrations at 1 and 3 min, increased with age and was significantly lower in ICA-negative siblings (86 +/- 6 microU/ml, P < 0.002) than in normal controls (115 +/- 6 microU/ml). This difference was not apparent before 8 yr of age. None of the ICA-negative siblings developed diabetes after an average of 4.5 yr. ICA-positive siblings at first study had a first phase insulin response similar to that of ICA negative siblings, but significantly lower than that of the normal controls (74 +/- 13 microU/ml, P < 0.02). The reason for the decreased insulin secretion in ICA-negative siblings is unknown, but could involve a defect in the growth of beta-cell mass or insulin secretion that could be part of the multifactorial pathogenesis of type 1 diabetes.

DECREASED INSULIN RESPONSE TO GLUCOSE IN ISLET CELL ANTIBODY-NEGATIVE SIBLINGS OF TYPE I DIABETIC CHILDREN

In order to determine immune and possibly non immune factors determining first phase insulin response to glucose (FPIRG) in siblings of type I diabetic patients, we performed a simplified intravenous glucose tolerance test in 170 normal children, 98 ICA negative and 12 ICA positive siblings of type I diabetic children. Normal children (age 3-16, 9.8±0.3 years) were patients hospitalized for reasons not interferring with glucose homeostasis and investigators' and colleagues' children. ICA negative siblings were similar to controls in respect to age (2-16, 9.3±0.4 years) and body mass index. Insulin was measured 1 and 3 minutes after i.v. injection of 0.5 mg/kg b.w. of 50% dextrose in 2.5 minutes. In normal children, FPIRG (1 + 3 minutes insulin measurements) increased with age, giving a linear regression curve of insulin 1 + 3 = 10.5*age + 11.3 (r=0.45). In comparison ICA negative siblings had a significantly lower FPIRG (86±6 vs 115±6 μU/ml, p<0.005). This difference was distributed evenly throughout the age range giving a linear regression curve of: insulin 1+3 = 4.6*age + 41 (r=0.3). None of the ICA negative siblings developped diabetes after a follow up of 4.5±1.1 years. ICA positive (≥20 JDF units) siblings had a FPIRG at first testing comprarable to that of ICA negative siblings (74±13 μU/ml, p<0.02 vs controls). Two of them became diabetic. We conclude that ICA negative siblings of diabetic patients have a decreased FPIRG. Further investigations are needed to elucidate the basis for this difference.

Sibling visitation: how far should the pendulum swing?

This study describes development of early interaction between siblings. Subjects (N = 22) were the older siblings (30 to 52 months) of healthy new babies. Data were collected to describe initial contact, regressive behaviors, and extent and quality of sibling interaction at 3 weeks, 5 months, and 7 months. Patterns of visualization, touch, and communication were observed. There was no evidence of sibling regressive behavior. Behaviors were more positive than negative and older siblings were in proximity for initiation of interaction by the new baby at 5 and 7 months. Findings indicate positive sibling relationships may be the norm rather than the exception.

Glutamic acid decarboxylase (GAD) autoantibodies are additional predictive markers of type 1 (insulin-dependent) diabetes mellitus in high risk individuals.

The prevalence of glutamic acid decarboxylase autoantibodies was determined with an immunotrapping enzyme activity assay in newly-diagnosed Type 1 (insulin-dependent) diabetic patients as well as in first-degree relatives using rat brain homogenate as a source of glutamate decarboxylase. Twenty-six out of 86 islet-cell cytoplasmic auto-antibody positive and one out of 24 islet cell autoantibody negative patients of recent onset, had autoantibodies to glutamate decarboxylase above the upper 99% confidence limit obtained from 89 control sera. Among 27 islet cell autoantibody positive relatives including 19 siblings and 8 parents, antibodies to glutamate decarboxylase were found in 8 of 9 (89%) relatives and 7 of 8 (87.5%) siblings with islet cell auto-antibody titres above 20 JDF units, in 1 of 19 (5.2%) relatives with islet cell autoantibody titres between 2 and 5 JDF units, in 2 of 263 (0.7%) siblings and 1 of 139 parents without islet cell autoantibodies. In first-degree relatives, high titre islet cell autoantibodies and autoantibodies to glutamate decarboxylase were tightly associated (X2 = 182, p = 0.0001). None of the relatives with low genetic risk (n = 64), i.e. HLA-different to the diabetic proband, was found to be antibody positive. Antibodies to glutamate decarboxylase were present only in those relatives sharing at least one haplotype with the diabetic proband, including two islet cell autoantibody negative but HLA-identical siblings. Autoantibodies to glutamate decarboxylase were present in 7 of 9 (77%) relatives who developed the disease, including one islet cell autoantibody negative sibling.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose‐induced Insulin Response is Reduced and Proinsulin Response Increased in Healthy Siblings of Type 1 Diabetic Patients

Glucose-stimulated insulin and proinsulin responses, and insulin sensitivity, were studied in 30 HLA identical, 38 HLA haplo-identical, and 25 HLA non-identical, healthy islet-cell-antibody negative siblings of Type 1 diabetic patients. The results were compared with 41 age- and sex-matched healthy subjects with no diabetes in the family. The proinsulin-corrected insulin response to an intravenous glucose infusion test was significantly lower among siblings when insulin sensitivity was taken into account (1.65 (inter-quartile range 1.20-2.64) vs 2.18 (1.65-3.28) nmol mmol-1 min, p = 0.04). Proinsulin values were consistently higher among siblings than among control subjects (peak values 50.0 vs 38.0 pmol l-1 (p = 0.004)). When proinsulin release was corrected for individual insulin sensitivity this difference remained. The results suggest disturbed islet B-cell function, unrelated to HLA identity or the presence of circulating islet cell antibodies.

Helicobacter pylori in children with peptic ulcer and their families

Little is known about the source and spread of Helicobacter pylori, but transmission from infected family contacts has been suggested. We have therefore investigated 15 children with peptic ulcer and their first-degree relatives for H. pylori. Serum anti-H. pylori IgG, pepsinogen I, and gastrin levels were measured. Endoscopy was carried out on the children and relatives, and biopsies were taken from the gastric antrum for histology, microbiology, and urease testing. Six of 11 children with duodenal ulcer (55%) and two of four children with gastric ulcer (50%) were positive for H. pylori. Fourteen of 16 parents (87%) and eight of 13 siblings (61%) of H. pylori-positive children with peptic ulcer were also infected compared with eight of 14 parents (57%) and none of four siblings of H. pylori-negative children with peptic ulcer (P less than 0.10, greater than 0.05, and NS, respectively). The children with H. pylori-negative peptic ulcer and negative siblings combined were younger than positive children with peptic ulcer and positive siblings (P less than 0.001). The reliability of serum anti-H. pylori IgG level as a screening test for infection was confirmed. These findings call into question a pathogenetic role for H. pylori in some childhood peptic ulceration, but do suggest that person-to-person spread of infection occurs.

Susceptibility of HLA-B27 transgenic mice to Yersinia enterocolitica infection

The majority of patients with reactive arthritis have the major histocompatibility complex class I gene HLA-B27. The development of arthritis in these patients often occurs following infection with one of several enteric bacteria, including Yersinia enterocolitica. In this study, transgenic mice expressing HLA-B27 and their negative full sibs were infected intravenously with Yersinia enterocolitica 0:8 WA in an attempt to develop an experimental model of reactive arthritis. To date, no reactive arthritis has been observed; however, a significantly higher incidence of paralysis was observed in the HLA-B27 + transgenic mice. Injection of 10 5 organisms induced hind limb paralysis in 8 out of 30 of the HLA-B27 transgenic mice (27%) and in only 1 of the 24 negative siblings (4%). Paralysis occurred in 14 out of 30 HLA-B27 + mice (47%) at a dose of 10 4 organisms. Only 2 of the 25 negative siblings (8%) were affected at this dose. Paraspinal abscesses were found in all of the paralyzed animals. At the 10 4 dose most of the HLA-B27 + mice (70%) succumbled to the disease within 4 weeks, while the mortality in their B27 − full sibs was less than 10%. Thus, HLA-B27 transgenic mice have higher mortality and morbidity from infection with Y. enterocolitica 0:8 WA than corresponding HLA-B27 − littermates.

Favourable outcome of patients older than 40 years of age after transplantation with marrow grafts depleted of lymphocytes by counterflow centrifugation

Nineteen patients with a median age of 43 years (range 40-48) were transplanted for acute myelogenous leukaemia (AML), refractory anaemia with excess of blasts in transformation (RAEBt), acute lymphoblastic leukaemia (ALL) in first complete remission, multiple myeloma, and chronic myelogenous leukaemia (CML) in chronic or accelerated phase. Their outcome was compared with that of 35 patients with a median age of 34 years (range 30-39), and a group of patients with age younger than 30 years (median 24; range 16-29) transplanted for the same indications. Donors were human leucocyte antigen (HLA)-identical, mixed lymphocyte culture (MLC) negative siblings. All marrow grafts were depleted of lymphocytes by counterflow centrifugation. The estimated event-free survival at 3 years after allogeneic bone marrow transplantation was 60.7% for patients with age greater than 39 years, 57.8% for patients with age less than 30, and 43.0% for the intermediate age group (P greater than 0.3). The estimated transplant-related mortality showed no tendency to increase with older age of recipients. The incidence of acute GVHD greater than grade 1 was 15.7% in patients with age greater than 39 years, 9.5% in patients younger than 30 years, and 23% in the intermediate age group. The incidence of chronic GVHD was higher in the older patients (39% compared to 24% in patients younger than 30 years, 19% in the intermediate age group). Chronic GVHD resolved completely in five out of seven patients aged 40 years or more. Reduction of the incidence of acute graft-versus-host disease by physical lymphocyte depletion allows allogeneic bone marrow transplantation for patients aged 40-50 years without increase of transplant-related mortality resulting in similar event-free survival in patients older than 40 years compared to those younger than 40 years.

113 RELATIONSHIP BETWEEN BASAL AND STIMULATED INSULIN RESPONSES AND INSULIN-LIKE GROWTH FACTOR 4 (IGF-1) IN NORMAL SUBJECTS AGED 5 TO 50 YEARS

Insulin and IGF-1 are structurally very similar and there are many functional links between the two hormone systems. However normal relationships between insulin and IGF-1 have never been defined. We performed intravenous glucose tolerance tests (0.5g/g/ maximum 25g) on 59 islet-cell antibody negative siblings of diabetic children and on 43 adults (aged 5.6-50 years). Puberty was staged (Tanner) and subjects divided into 4 groups: I - stage 1 (n=22), II - stages 2 & 3 (n=17). III - stages 4 & 5 (n=20), IV - adults >17 years (n=43). The pattern of fasting IGF-1 concentrations, fasting insulin and incremental 0-60 minute insulin areas (following IV glucose) in Groups I to IV was markedly similar levels rose significantly throughout puberty (p<0.001 for all parameters) and declined to prepubertal levels by the third decade. IGF-1 concentrations: I - 0.85±0.44, II - 1.72±0.81, III 2.35±0.83, IV - 1.11±0.51 U/ml. Fasting insulin concentrations: I - 7.5±3.2, II - 13,6±5.0, III - 13.8±3.3, IV - 7.6±2.9 mU/l. Incremental 0-60 minute areas: I - 1163±433, II - 2047±668, III - 2441±675, IV - 1117±485 mU/l/min. There was a strong positive correlation between logged insulin and logged IGF-1 levels; fasting insulin versus IGF-1 concentrations r=0.625, p<0.001 incremental 0-60 min areas versus IGF-1 concentrations r=0.572 p<0.001. This relationship, constant from 5 to 50 years, suggests that insulin may have an important influence on growth during childhood.

The pattern of basal and stimulated insulin responses to intravenous glucose in first degree relatives of type 1 (insulin-dependent) diabetic children and unrelated adults aged 5 to 50 years.

The pattern of insulin secretion was studied in 107 normal individuals aged 5 to 50 years. Intravenous glucose tolerance tests were performed on 64 islet-cell antibody negative siblings of diabetic children and on 43 normal adults. Puberty was staged using Tanner's criteria and subjects were grouped as follows: I - stage 1 (n = 22), II - stages 2 and 3 (n = 18), III - stages 4 and 5 (n = 20), IV - adults greater than 17 years (n = 47). Basal and stimulated (incremental 0-10 and 10-60 min areas) insulin responses rose throughout puberty (Groups I-III), declined following puberty until the third decade (Groups III and IV) and then appeared constant thereafter. Insulin levels in the 17.6-22.5 year group were lower than in the 12.6-15 year group (p less than 0.01). Fasting insulin to glucose ratios and incremental 0-60 min insulin to glucose area ratios produced a similar age-related pattern indicating that changes in insulin levels were independent of glucose concentrations. Gender did not affect these changes and multiple regression analysis showed that HLA haplotype sharing did not influence insulin responses in siblings of diabetic patients. Age and pubertal status must be carefully considered when interpreting intravenous glucose tolerance tests from patients suspected of having early abnormalities of carbohydrate metabolism.

Basal and stimulated insulin levels rise with advancing puberty.

We studied the effect of pubertal development on insulin secretion. Intravenous glucose tolerance tests were performed on 47 islet-cell antibody negative siblings of diabetic children and on 16 normal adults. Puberty was staged using Tanner's criteria and subjects were grouped as follows: I, stage 1 (n = 16); II, stages 2 and 3 (n = 15); III, stages 4 and 5 (n = 16); IV, adults (n = 16). Fasting insulin increased with advancing puberty (p = 0.59, P less than 0.001). The stimulated insulin response also rose with increasing pubertal development: for the 0-10 min insulin area, p = 0.46, P less than 0.001 and for the 10-60 min area, p = 0.68, P less than 0.001. There was a low positive correlation between insulin and age to 16 years but multiple regression analysis showed that this could be accounted for by puberty alone. Indeed prepubertal children and adults did not differ. There was no correlation between glucose (fasting and 0-60 min area) and puberty or age. These findings suggest that insulin resistance increases during puberty, and this may contribute to the frequency of presentation or worsening control of insulin dependent diabetes at this time.

A possible predictive test for graft-versus-host disease in bone marrow graft recipients: the mixed-epidermal cell-lymphocyte reaction.

The possible ability of the mixed-epidermal-cell-lymphocyte reaction (MECLR) to detect alloreactivities in HLA-identical MLR negative siblings has been investigated before grafting in 30 donor-recipients pairs and their families. Results indicate that recipients' epidermal cells (EC) can induce proliferative responses of HLA-identical MLR-negative donors' lymphocytes in 55% of the pairs tested. Moreover, further evaluation of 21 patients shows that the positivity of the MECLR before the graft is correlated with later appearance of acute and chronic GVHD, and especially with the severity of cutaneous injury. EC have been shown to be more efficient antigen presenting cells than peripheral blood lymphocytes for in vitro primary proliferative responses, so these reactions could be directed against some minor histocompatibility antigens, thus leading to possible improvement in selecting bone marrow graft donors and to the detection of donor-recipient pairs with a high risk of GVHD.

Standard conditioning regimen and t-depleted donor bone marrow for transplantation in chronic myeloid leukemia

Between January 1984 to June 1985, 18 Ph 1 positive chronic myeloid leukemia (CML) patients in chronic phase (CP) underwent allogeneic bone marrow transplantation (BMT) from HLA identical and MLC negative siblings. The median age was 32.5 yr and median disease duration of CML at time of BMT was 19.3 months. The pretransplant conditioning regimen consisted of cyclophosphamide (CTX) (120 mg/kg) and 10.20 Gy total body irradiation (TBI) at 6 doses of 1.7 Gy each, administered in 3 daily fractions over 2 days at a dose rate of 15–20 cGy/min. To prevent graft-vs-host disease (GvHD) we used methotrexate (MTX) in one patient and cyclosporin-A (CYA) in the other 17 patients. In addition to CYA, given until day +365, 10 patients received donor marrow depleted of T cells with CAMPATH-1. The residual marrow lymphocytes were always <1%. The rate of engraftment was significantly correlated with the number of nucleated cells infused. Neither GvHD nor graft failure were observed among CAMPATH-1 patients. In this group one cytogenetic and one hematologic relapse occurred. The overall actuarial survival at 24 months is 78%. Of the 10 patients treated with donor marrow depleted of T cells, 9 are alive after a median follow-up of 9 months (range 5–18), with an actuarial survival of 90%. Of the other 8 patients transplanted with untreated marrow, 5 are alive after a median followup of 19.3 months (range 3.7–24) and the actuarial survival is 63.8%. This pilot study seems to demonstrate that T-cell depletion of donor bone marrow with CAMPATH-1 is effective to prevent GvHD, while the risk of graft failure can be avoided using a “standard” conditioning regimen including a fractionated TBI with a fast dose rate and a prolonged administration of CYA at the maximum tolerable dosage. While the high frequency of relapses suggests the employ of more aggressive anti-leukemic conditioning regimens in CAMPATH-1 treated marrow recipients.

Human epidermal cells are more potent than peripheral blood mononuclear cells for the detection of weak allogeneic or virus-specific primary responses in vitro

Human epidermal cells (EC) and peripheral blood mononuclear cells (PBMC) have been used as antigen-presenting cells in allogeneic reactions or in self-restricted antiviral responses. Comparison of results from both cell types indicates that: (1) EC were better stimulators of primary proliferative responses in all the antigenic systems tested. (2) In secondary reactions, EC and PBMC functioned similarly for allogeneic responses, while a weak but significant difference could be observed in both (HSV1 or influenza A) virus-specific reactions. (3) By comparing pairs of HLA-identical mixed lymphocyte reaction (MLR)-negative siblings, positive responses were observed in several different families when lymphocytes of potential bone marrow donors were stimulated by EC of the recipient. This suggests that EC might be useful in detecting relatively weak proliferative responses in a number of antigenic systems, but especially in primary reactions against viral or putative minor histocompatibility antigens. (4) Despite this stronger antigen-presenting capacity in proliferative responses, EC induced lower levels of cytotoxic T lymphocyte (CTL) reactions than PBMC, not only in allogeneic responses but also in virus-specific self-restricted reactions.

Bone marrow transplantation in children with aplastic anemia and acute lymphatic leukemia.

A boy with severe Aplastic Anemia (AA) and a girl with Acute Lymphoblastic Leukemia (ALL) in relapse have been grafted with marrow from HL-A identical, mixed leukocyte culture (MLC) negative siblings after appropriate immunosuppressive and antileukemic therapy. Both of them are well 7 and 2 months after transplantation respectively. Bone marrow transplantation should be considered in children with AA and ALL in relapse, if HL-A identical, MLC negative siblings are available.

Report on a family with six Tjanegative siblings.

A pregnant woman for whom it was not possible to find compatible blood needed for transfusion, was found to have the rare blood type pp (Tja‐negative) with anti‐P + P1 antibodies in her serum. The patient was one of ten living sibs among whom four brothers and one sister had the same rare type as the proposita. Results of genetic and immunohematologic studies on this sibship and their parents were presented. The obstetric history of the proposita included three miscarriages. The fourth pregnancy, terminated by cesarean section, resulted in a premature infant, whose death was attributed to hyaline membrane disease. The fifth pregnancy ended with a full‐term normal infant who remained free of disease. Positive direct antiglobulin tests were obtained with cord blood specimens of both infants, indicative of transplacental transfer of the maternal anti‐P + P1 antibodies. However, manifest hemolytic disease of the newborn did not develop in either infant. The observations made in this study were compared with data recorded in the literature on this subject and discussed in relation to clinical and laboratory problems arising from the presence of the rare blood type p.