Frank B. Gelder

Correlation between sex hormone binding and peritumoral edema in intracranial meningiomas.

Estrogen and progesterone receptor binding activity was measured in 22 intracranial meningioma surgical specimens. None of the tumors was estrogen receptor-positive, whereas 19 were progesterone receptor-positive. Of these 19 patients, all demonstrated significant computed tomographic (CT) evidence of peritumoral edema. None of the 3 patients who lacked progesterone receptor binding had CT evidence of peritumoral edema (P less than 0.005). Peritumoral edema associated with intracranial meningiomas seems to be related, at least in part, to progesterone binding activity. This implicates the potential use of progesterone antagonists for the treatment of incompletely resected or recurrent meningiomas.

Detection of panel-reactive anti-HLA class I antibodies by enzyme-linked immunosorbent assay or lymphocytotoxicity: Results of a blinded, controlled multicenter study

A soluble HLA ELISA for the detection of anti-HLA class I IgG antibodies was developed and compared to complement-dependent microlymphocytotoxicity. ELISA plates were coated with a panel of sHLA class I antigens isolated from the culture supernatants of 46 different EBV-transformed phenotyped B-cell lines. After the incubation of the coated plates with test serum, bound antibodies were detected using a peroxidase-conjugated anti-human IgG antibody. Absorbance was read using an ELISA plate reader and assay results were analyzed by computer. Antibody specificities were determined by Fishers exact test tail analysis. The reproducibility of ELISA assay results was evaluated in a blinded, controlled multicenter study. A total of 102 serum specimens from patients on waiting lists to receive kidney transplants were tested five times by ELISA in five different laboratories. The correlation coefficients (r) of %PRA values determined by ELISA ranged from 0.89 to 0.96, and the average agreement on qualitative assay results (antibody positive vs antibody negative) was 98%. Endpoint titration of several serum specimens demonstrated equivalent sensitivity of ELISA and microlymphocytotoxicity (using the anti-globulin antibody protocol). Most of the antibody specificities determined by ELISA were in agreement with specificities determined by microlymphocytotoxicity. To evaluate the correlation of ELISA and microlymphocytotoxicity (CDC) assay results the same 102 specimens were tested six times by CDC in five different laboratories. The interlaboratory correlation coefficient (r) of %PRA values determined by microlymphocytotoxicity ranged from 0.57 to 0.94, and the average agreement on qualitative assay results was 85%. A comparison of ELISA with microlymphocytotoxicity was performed using consensus microlymphocytotoxicity results. This showed a high correlation (r = 0.81) of %PRA values determined by ELISA and microlymphocytotoxicity. This demonstrates that the detection of anti-HLA class I antibodies by soluble HLA ELISA is a reliable alternative to microlymphocytotoxicity testing.

Soluble HLA class I in the serum of transplant recipients.

BACKGROUND Soluble HLA, Class I (S-HLA-I) has been found in serum, plasma, body fluids, peritoneal dialysates, and urine. S-HLA-I may be a product of membrane shedding, proteolysis, and/or alternate gene splicing. Previous assays to quantitate S-HLA-I were cumbersome, required radioisotope labeling procedures, or the purification of Class I antigen preceding antigen quantitation. The authors developed a solid-phase, enzyme-linked immunoassay that can be used to quantitate S-HLA-I and to study its relevance in transplantation. METHODS A solid-phase enzyme-linked immunoassay employing monoclonal anti-Class I to catch S-HLA-I present in plasma and peroxidase-labeled monoclonal anti-beta 2-microglobulin (B2M) to quantitate bound S-HLA-I was employed. Values were correlated with rejection and infection episodes. Pre and postoperative determinations were made from the sera of liver, heart, and kidney recipients. Size chromatography was used to compare the molecular weight of S-HLA-I from baseline and peak serum concentrations obtained during rejection episodes (2 liver, 1 heart, 1 kidney), and from 1 kidney recipient with a wound infection. RESULTS All 9 liver recipients and 12 heart recipients demonstrated a fall in S-HLA-I, or very low initial values, for the first 10 days and then a progressive increase in values substantially above preoperative concentrations. Values from renal recipients were more variable. There were temporary increases in S-HLA-I preceding or during 16 of 20 (80%) biopsy-proven rejections (all reversible), and in 9 of 11 (83%) episodes of infection (bacterial, viral, and fungal). In heart and liver rejection, as well as the wound infection, the sera contained increased S-HLA-I, which was almost all of the same molecular weight (approximately 52,000 daltons). In serum from the one patient with renal rejection, two additional S-HLA-I peaks occurred, one with a molecular weight near 1,000,000 daltons and the second at a molecular weight approximately 11,000 daltons suggesting cellular breakdown of the donor organ. CONCLUSION In summary, different patterns of S-HLA-I concentrations occur after kidney transplantation. Most liver and heart recipients reached a steady state higher than preoperative levels. Transient increases in S-HLA-I occurred with rejection and infection. In one severe rejection episode, larger and smaller fractions of S-HLA-I were detected and may represent cell membrane breakdown.

Soluble HLA antigens and ELISA--a new technology for crossmatch testing.

A soluble HLA ELISA for the detection of donor specific anti-HLA class I IgG antibodies was developed and compared with microlymphocytotoxicity. Donor sHLA was prepared from donor blood or purified blood lymphocytes and captured onto monoclonal antibody coated ELISA plates. After incubation of captured HLA with test serum, bound IgG antibodies were detected using a peroxidase-conjugated anti-human IgG antibody. Serum samples from patients on waiting lists to receive kidney transplants were tested by lymphocytotoxicity (AHG protocol) and/or sHLA ELISA in four different laboratories using HLA preparations from eight organ donors. Concordant crossmatch results were obtained for 854 (99%) of 864 ELISA crossmatches. In contrast, concordant results were obtained for 234 (91%) of 256 lymphocytotoxicity crossmatches. Interlaboratory reproducibility of ELISA results was 99%. In contrast, interlaboratory reproducibility of lymphocytotoxicity assay results was 78%. Endpoint titrations of serum specimens containing anti-HLA antibodies demonstrated equivalent sensitivity of ELISA and AHG lymphocytotoxicity crossmatch and similar sensitivity of ELISA and flow cytometry crossmatch. Specimens tested positive by lymphocytotoxicity without DTT treatment but negative with DTT treatment were tested negative by ELISA. Comparison of lymphocytotoxicity and ELISA crossmatch results showed an agreement of 94%. This demonstrates that detection of anti-donor HLA class I antibodies by ELISA is a reliable alternative to microlymphocytotoxicity testing.

Soluble HLA in human body fluids

There is a growing body of information about the soluble forms of HLA in serum but there are only a few reports discussing sHLA in other body fluids. We quantitated sHLA-I and sHLA-II concentrations in sweat, saliva and tear samples from five normal individuals with known HLA-phenotypes. We also studied sweat samples from an additional 12 normal nonphenotyped subjects, as well as in CSF of 20 subjects with different illnesses, using solid phase enzyme linked immunoassay. Sweat, saliva and tears from normal subjects were found to contain very low or nondetectable amounts of sHLA-I. In contrast, sHLA-II molecules were found in each of these body fluids, although, with considerable variation between individuals. The presence of sHLA-II in saliva was further confirmed by Western-blotting. It was observed that sHLA-II having molecular mass of 43,900 and 18,100 daltons was comparable with that found in serum from normal individuals. In addition, no association of sHLA-II levels with allospecificities in either body fluid or in serum was apparent. The results of CSF sHLA concentrations in different diseases were as follows: (1) High CSF SHLA-I levels were measured during viral encephylitis (n = 3), while none of these patients contained sHLA-II in CSF; (2) The levels of sHLA-II, but not sHLA-I were elevated in CSF of patients during seizure (n = 6) and of patients with neonatal hepatitis (1 of 2) or with connective tissue disease accompanied with viral infection (n = 2); (3) No CSF sHLA-I or sHLA-II could be detected at polyneuropathy (n = 2), or in patients with syphilis (n = 3), or leukemia (n = 2) with evidence of neurologic involvement of central nervous system. Taken together, it may be concluded that the presence of sHLA in several body fluids is physiologically normal. It appears that sHLA-II is the predominant class of HLA molecules present in different body fluids. We propose that the system responsible for sHLA-II production in various body fluids must involve different mechanisms than those responsible for sHLA-I synthesis in serum.

Soluble HLA class II concentrations in normal individuals and transplant recipients. Comparison with soluble HLA class I concentrations.

We developed an ELISA to quantify soluble HLA class II (S-HLA-II) in 702 sera obtained from normal subjects, patients with end-stage renal disease, and recipients of renal, hepatic, and cardiac transplants. Concentrations of S-HLA-II were detectable in 124 of 126 normal individuals. The distribution of normal values described a monophasic curve with a skewed distribution. In transplant recipients, there were no differences between preoperative and posttransplant values, but values in liver patients were significantly higher than in kidney patients, and values for heart patients were lowest of all groups. There were periodic variations in concentrations in individual patients, but these were unrelated to rejection, infection, or any other apparent clinical event. S-HLA-II was consistently present in the urine. All of these observations contrast with previous observations concerning soluble HLA class I (S-HLA-I) molecules, which were almost the precise reverse. It seems likely that these clear differences in S-HLA-II and S-HLA-I concentrations relate to different physiologic processes in either production, function, or elimination.

SOLUBLE HLA-I IN RHEUMATIC DISEASES

OBJECTIVE To study serum levels of Class I soluble HLA (sHLA-I) in patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), polymyositis or dermatomyositis (PM/DM) or scleroderma and to assess the possible influence of ethnic factors on concentration in each disease group. METHODS Solid-phase enzyme linked immunoassay was used to measure sHLA-I in the serum of 385 patients with varied ethnic backgrounds (American-Caucasians, African-Americans, Georgian-Caucasians) with rheumatic diseases. Studies on patients were compared to similar measurements of 189 healthy individuals. RESULTS Mean sHLA-I levels were significantly higher in patients with SLE than those observed in healthy individuals or other rheumatic diseases. Highest concentrations were present in Georgian-Caucasian patients with SLE. American-Caucasian patients with RA or scleroderma had higher sHLA-I levels than normal Caucasian individuals. The majority of patients with PM/DM in all ethnic subgroups were low secretors of sHLA-I. CONCLUSION Mechanisms underlying the secretion of sHLA-I appear to differ among the rheumatic diseases studied and various ethnic groups. These genetic differences in sHLA-I secretion could be associated with ethnic and pathophysiologic differences among these rheumatic diseases.

Tumor-associated antigens are cytokine inducers and hyporeactivity factors to the immune system

We investigated possible mechanisms leading to the inhibition of theimmune system in people with chronic disorders. Tumor cell produce proteinreleased into the circulation, such as tumor associated antigens, may playan important role in processes preceding paralysis of the immune system. Totest this hypothesis the following tumor associated antigens were used: AFP,OFP, CA-125, CA-50 and CA-19-9. Their role was assessed by modulatingcytokine production in cord blood lymphocytes and peripheral white bloodcells obtained from grown population of patients treated with colostrinin,an cytokine inducer. PHA, LPS and colostrinin were used as positive controlin those essays. Each antigen tested individually induced IFN, TNFαand IL-6 in dose dependent fashion. None of the tested cytokines werespontaneously released by the cells. Data generated from these experimentsindicated that tumor associated antigens are inducing type 1 cytokines insimilar fashion as LPS or colostrinin. However, lymphocytes taken frompatients undergoing therapy with colostrinin revealed progressive losscapability to produce type 1 cytokines as they did in case of colostrinin.The loss of the capability to respond to antigen may represent phenomenonleading to immune tolerance.

Soluble HLA Class I Antigens in Patients with Type I Diabetes and Their Family Members

Our objective was to study a possible contribution of MHC genes to S-HLA-I secretion in patients with Type I diabetes. Quantitatively, we used a highly sensitive enzyme-linked immunoassay to measure S-HLA-I in the serum of a total of 39 patients with Type I diabetes, as well as 36 kinships of 12 diabetic patients and 82 normal individuals with known HLA-phenotypes. S-HLA-I levels were abnormally elevated in patients or their non-diabetic relatives compared to normal controls (p < 0.0009). No complete HLA-haplotype had been identified to be correlated with high or low S-HLA-I secretion. Only the HLA-A23 or A24 (splits of HLA-A9) positive individuals sera were found to contain high S-HLA-I concentrations in all populations studied. The difference in S-HLA-I levels of HLA-A24 patients (n = 4) or their HLA-A24 positive non-diabetic relatives (n = 10) to the group of HLA-A24 normal controls (n = 15) was statistically highly significant (p < 0.0005 and p < 0.0009, respectively). The results suggests that HLA-A24 may confer additional independent risk for the disease expression in male children but not in female siblings. Nevertheless, the data implies that the patients or their non-diabetic relatives carrying the HLA-A24 have increased risk of developing ICA associated with high S-HLA-I levels compared to HLA-A24 negative probands or their kinships with low levels of S-HLA-I. This effect occurred irrespective to other diabetes related HLA-DR alleles. In summary, the results show a pronounced genetic heterogeneity of Type I diabetes with MHC control of the expression of S-HLA-I and possible involvement of hormonal factors that might potentiate a specific synthesis of S-HLA-I. The findings have implications for identifying individuals with a possible risk for developing the disease.

Serologic allogeneic chimerism.

BACKGROUND At least some transplanted livers secrete soluble human leukocyte antigens (sHLA) of donor phenotype into the body fluids of recipients. The individuals in whom this phenomenon occurs are by definition serologic allogeneic chimeras. Because an allogeneic transplanted liver may induce tolerance to itself and other organs in animals of the donor strain, and because maintenance of a soluble antigen in the circulation of any animal in sufficient quantity for a sufficient period generally leads to tolerance, this phenomenon may be biologically important. This study was performed to determine how common this phenomenon is and whether it occurs after transplantation of organs other than the liver. METHODS We studied 445 serum samples obtained from transplant recipients (liver, n=12; kidney, n=18; and heart, n=8) before and at various intervals after transplantation. All patients studied had allografts that had functioned for more than 1 year. We used an enzyme-linked immunosorbent assay to quantitate sHLA-A2 and sHLA-A1/A3/A11 (as a cross-reacting group). Donor and recipient combinations were selected in which measurable allotypes in donors were not present in recipients. In some instances, an additional allotype was present in a recipient but not in a donor. RESULTS All liver transplant recipients had detectable donor sHLA in their serum samples after transplantation. In 72% of kidney and 50% of heart transplant recipients, donor sHLA was found persistently in serum samples obtained after transplantation. Interestingly, all heart transplant recipients of HLA-A3, but none of HLA-A2, had detectable donor sHLA in their serum samples, a finding that may be due to technical reasons. High and stable serum concentrations of donor sHLA characterize long-term stable allograft function. CONCLUSIONS Donor sHLA is produced by all transplanted livers, most transplanted kidneys, and at least half of (but probably more) transplanted hearts. The hypothesis that donor sHLA may be tolerogenic to liver transplants can be expanded to include kidney and heart transplants.