Beta-2 microglobulin-free HLA class I heavy chain (FHC) A3 and/or A30 soluble products contribute only minimally to serum FHC expression.

No monoclonal antibodies (mAbs) are presently available to measure the total amount of beta2-microglobulin-free HLA class I heavy chain (FHC) in sera. The available ELISA-based double determinant immunoassay (DDIA), established to measure FHC, uses two mAbs (TP25.99 and HC-10) that recognize a monomorphic determinant expressed on all HLA-B/C FHC products and a determinant expressed only on some HLA-A FHC products. This restricted reactivity implies that, in addition to HLA-B/C, HLA-A FHC products are also detected in individuals bearing HLA A3 and/or A30 allotypes. The aim of this study was to establish whether such restriction results in the detection of low FHC levels in individuals lacking HLA A3 and/or A30 allospecificities. The FHC mean concentration (+/- SD) in 294 healthy blood/bone marrow donors (HBDs) was 0.24 (+/- 0.2) mg/l. The grouping of HBDs according to their HLA-A FHC product reactivity with one, both or no mAbs did not result in any statistically significant differences (Mann-Whitney test: P > 0.05) between their median FHC concentrations. Since the absence of differences in their FHC levels was not attributable to a difference in the percentage distribution of HLA allotypes associated with high or low HLA-B/C FHC expression, our results indicate that FHC HLA A3 and/or A30 products detected in DDIA by these two mAbs only minimally contribute to FHC serum expression and that the assay is not limited by the failure to detect HLA-A FHC products in A3- and/or A30- individuals.

Human CD4 internal antigen anti-idiotypic monoclonal antibody. immunochemical and sequence analysis.

The mouse mAb2 16D7 recognizes the paratope of the syngeneic anti-human CD4 mAb HP2/6 (mAb1 of our idiotypic cascade) and mimics CD4 in xenogeneic settings in humans. Immunochemical and sequence analyses were performed to define the minimum structural requirement for this mimicry. Binding assay of mAb1 with isolated naive 16D7 H and L chains showed that only the second reacted with mAb1. Specificity was indicated by the lack of reactivity of mAb1 with the L chain of mAb2 14D6, which also recognizes mAb1-paratope. It is likely that the 16D7-L mAb1-specific epitope is "sequence-dependent", since fully denatured 16D7-L still reacted with mAb1. Sequence analysis of 16D7 and mAb1 showed a high degree of homology of their VH. as both were coded by the same gene family (V/II), whereas CDR3 showed the greatest diversity. Alignment of 16D7-H CDR3 with CD4, however, produced no similarity. In contrast, analyses of the 16D7 VL sequence (XX/V) defined a CDR3 6-mer peptide with a 50% identity (83% of similarity) to the CD4 stretch 218-223. This peptide seems a suitable replacement for 16D7 in active immunotherapy as it did not match any protein fragment retrieved from the n-r database (NCBI) and both the peptide and the corresponding CD4 amino acid stretch are surface accessible. Based on their immunochemical profiles and similarity to CD4, four additional 16D7-derived peptides were designed for synthesis. The data indicate that CD4 mimicry by mAb2 can be obtained at the level of primary structure and provide useful information for the synthesis of peptide(s) with bioactive potential.

Monoclonal antibodies in the immunotherapy of autoimmune diseases.

The present report critically reviews the rationale, clinical effectiveness and limits of monoclonal antibody-based immunotherapy in the treatment of autoimmune diseases, with particular emphasis on tumor necrosis factor-alpha blocking reagents. Reference will also be made to active immunotherapy whereby an endogenous response induced by anti-idiotypic monoclonal antibodies or peptides toward molecules regarded as passive immunotherapy targets, is expected to mediate the therapeutic effects.

Serum levels of beta-2-microglobulin-free heavy chain of HLA class I antigen in healthy individuals: relationship to their class I allotype.

An ELISA-based double determinant immunoassay has been established to measure the soluble beta2-microglobulin (beta2m)-free heavy chain (FHC) of the HLA-B, -C (and HLA-A3, -A28 and -A30) class I molecular complex in sera from 212 HLA-typed healthy unrelated individuals. FHC was calculated by means of a standard curve constructed using serial concentrations of beta2m-associated HLA-class I heavy chain (HLA-I)/FHC purified from cultured human lymphoid cell C1R-sB7-supernatant. The mean FHC concentration (+/-SD) was 0.25 mg/l (+/-0.2). Its median concentration did not statistically differ between males and females, though the male/female ratio was greater in the high secretor (FHC >0.45 mg/l; mean + 1SD) than in the low secretor group (FHC < 0.05 mg/l; mean - 1SD). FHC < 0.05 mg/l was statistically (Fisher's exact test) associated with HLA-B17 (p = 0.003); FHC > 0.45 mg/l was statistically associated with HLA-B35 (p = 0.003) and -Cw4 (p = 0.002). None of these allele-positive groups showed a mean FHC concentration 1.5 times higher than that of the corresponding allele-negative ones. This allotype-dependent HLA-B and C FHC enhancement was less marked than that previously reported for HLA-I in individuals carrying HLA-A9 (and its splits). These results indicate that FHC could be a more valuable marker when its levels are compared among individuals carrying different allotypes. Moreover the lack of correlation between FHC and HLA-I levels measured in 52 HLA-A3, -A28 or -A30 positive individuals suggests that the two molecules may be regulated by different metabolic pathways and their serum expression may have a different biological significance.

Increased serum levels of beta2m-free HLA class I heavy chain in multiple myeloma.

Serum levels of beta2-microglobulin (beta2m)-free HLA class I heavy chain (FHC) in 94 patients with multiple myeloma (MM) were higher than in 29 patients with monoclonal gammopathy of undetermined significance (MGUS) (P = 0.023) and in 97 sex- and age-matched healthy controls (P < 0.0001). Spearman correlation analysis indicated that in MM, FHC correlated with beta2m (r = 0.31, P = 0. 003) and the percentage of bone marrow plasma cells (BMPC%) (r = 0. 36, P = 0.002), whereas beta2m, in addition to BMPC% (r = 0.43, P = 0.0003), also correlated with creatinine levels (r = 0.63, P < 0.0001), haemoglobin levels (r = -0.35, P = 0.0007) and patient age (r = 0.34, P < 0.0011). Furthermore, MM patients with poor prognosis (beta2m >/= 6 mg/l) displayed higher FHC levels than those with a better prognosis (beta2m < 6mg/l) (P < 0.021). At variance from beta2m, these levels were not influenced by renal failure, as indicated by the lack of Spearman correlation of FHC with creatinine concentration and of statistical significance between the median FHC concentration of MM patients with creatinine < 176.6 micromol/l and those with creatinine >/= 176.6 micromol/l (P = 0.3). Stratification of patients according to disease activity and stage showed that FHC levels were only statistically different (P = 0.04) for disease activity, whereas beta2m and C-reactive protein were not. Taken together, our data indicate that serum FHC may be a useful disease marker in MM.

Artificial lamina-assisted laminoplasty performed in seven cases.

OBJECT: The authors attempted to simplify the operative approach to severe multilevel cervical spondylotic myelopathy. Seven patients with progressive and severe myelopathy underwent modified double-door laminoplasty during a 5-month period. METHODS: The double-door laminoplasty procedure was modified by using two artificial titanium laminae obtained by simple surgical 0.5-mm Ti-mesh (rather than by bone graft or ceramic spacers). Preoperatively, gait disturbance was present in all patients with long-tract signs on neurological examination. In all cases the sagittal diameter of the cervical spinal canal was somewhat reduced (< 10 mm) by congenital stenosis, and further severe compression of the spinal cord resulted from osteophytic bars and calcified ligamenta flava at different levels. No abnormal alignment, pathological movements, or instability was present. Computerized tomography (CT) studies demonstrated severe multilevel cervical compression, and T2-weighted magnetic resonance (MR) imaging demonstrated pathological areas of hyperintensity within the spinal cord in all cases. In the initial follow-up study (range 8-12 months), the patients who underwent this procedure experienced marked improvement of gait disturbance without any significant incidence of morbidity or complications. Postoperative CT and MR imaging studies demonstrated complete spinal cord decompression and restoration of the patency of the subarachnoid spaces. CONCLUSIONS: The proposed procedure has the advantage of achieving both an immediate stabilization of the open laminae by means of a bridgelike mechanism and protection from the possible compression of the dural sac by paravertebral muscles.

Evaluation of biotinylated cells as a source of antigens for characterization of their molecular profile.

Biotinylated lymphoid cells have been suggested as a useful source of antigen for the immunochemical characterization of their molecular profile. Labelling with biotin eliminates the problems associated with the use of radioactivity. However, this method has not been widely used. This reflects: (1) difficulties in optimizing the signal/background ratio because of the lack of a simple method to quantify biotinylated proteins in a cell lysate, (2) the loss of reactivity with monoclonal antibody of antigen following biotinylation, because of steric hindrance, and (3) the lack of information about the utility of other biotinylated cells as an antigen source. To overcome these limitations, we developed an ELISA to quantify biotinylated proteins in cell lysates and optimized the signal/background ratio. The validity of this approach was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of a number of cell surface antigens immunoprecipitated from lymphoid cells by an optimal amount of monoclonal antibody. Furthermore, we showed that biotinylated melanoma cells are a useful source of antigen for immunoprecipitation experiments and that ligation of biotin to antigen does not affect reactivity with monoclonal antibody. Lastly, biotinylated antigens in cell lysates stored at -80 degrees C for 6 months maintained their reactivity with monoclonal antibodies. Biotinylated cells thus represent a useful source of antigen for characterizing the immunochemical profile and analyzing the specificity of antibodies with immunochemical methods.

Absence of streptococcal protein G (PG)-specific determinant in the Fab region of human IgG2.

It has been previously reported that CH1 Fab protein G-contact site is responsible for the widespread recognition of mouse and human IgG Fab by PG. Here we present evidence that PG binding to F(ab')2 is restricted, as indicated by the lack of reactivity with PG-Sepharose columns of a portion of F(ab')2 fragments obtained by pepsin digestion of human IgG from a commercial immunoglobulin preparation for intravenous use or purified from sera of two healthy blood donors and two patients with polyclonal hypergammaglobulinaemia. Isoelectric focusing showed that F(ab')2 fragments that did not bind PG focused in a lower pH range compared with those which did. Testing of the Fab fractions with MoAbs to kappa and lambda light chains or to gamma1, gamma2 and gamma3-Fab subclass determinants showed that gamma2-F(ab')2 were mainly found in the PG non-reactive F(ab')2 fraction, and that this distribution was not influenced by the L chain isotype. These results indicate that the PG-specific binding determinant(s) is not expressed in the F(ab')2 region of most human IgG2.

The Fab region of IgG2 human myeloma proteins does not bear the streptococcal protein G-specific determinant.

Seven out of ten Fab (F(ab')2/Fab') preparations derived from purified human myeloma IgG showed a substantial binding to protein G-Sepharose. Subclass analysis revealed that the 7 protein G-reactive Fabs included 3 IgG1, 2 IgG3 and 2 IgG4 Fabs, whereas the remaining 3 which were not adsorbed were IgG2 Fab. Incubation of protein G-Sepharose with non-saturating amounts of 4 Fab preparations, representative of all IgG subclasses, showed that gamma 1, gamma 3 and gamma 4 Fabs adsorbed from 26 to 28.3%, whereas 80% of gamma 2 Fab was left in the supernatant after adsorption. These results indicate that human IgG2 lack PG-specific Fab-associated reactive site(s).

Anti-CD4 monoclonal antibody (mAb) and anti-idiotypic mAb to anti-CD4 in the therapy of autoimmune diseases.

The present report critically reviews the rationale, experimental and clinical effectiveness and limits of anti-CD4 monoclonal antibody (mAb) therapy. References are also made to a novel approach involving active immunotherapy and an anti-idiotypic mAb bearing the internal image of human CD4 antigen. Preliminary observations concerning the effects of this treatment in one patient with rheumatoid arthritis and in one patient with systemic lupus erythematosus are reported.

Natchev's auto-traction for lumbago-sciatica: effectiveness in lumbar disc herniation.

In Lind's auto-traction (LAT) for lumbago-sciatica, the patient provides traction force by pulling with the arms on a specially designed table, which also allows painless mobilization of the lumbar spine and passive traction. Two studies reported that one to 15 one-hour sessions on successive days might be sufficient to relieve pain in 25% to 90% of cases with verified lumbar disc herniation. Unfortunately, the technique imposes tiring manual efforts on the therapist and requires that the patient be transported by ambulance and confined to bed for a long time. These inconveniences were removed in a new version of the treatment proposed by Natchev. The effectiveness of Natchev's auto-traction (NAT) was evaluated in an open prospective trial on 77 patient with chronic lumbago-sciatica refractory to previous therapies, and herniation of one or more lumbar discs verified by computed tomography (CT) or myelography. Thirty-six of the 77 patients (47%) responded to the treatment in three to ten (median = 5) half-hour sessions. Pain intensity dropped to 27% (median) of the pretreatment intensity. Six months after treatment, 28 of the 36 responders were stable; only four had undergone surgery. By contrast, 20 of the 41 nonresponders had been operated on. The severity of either the radiologic or the neurologic picture was not predictive of the outcome. NAT was as effective as LAT: thus, due to its greater convenience it appears to be suitable as a routine approach in lumbar disc herniation and as a screening technique before surgery.

Microsurgical treatment of intramedullary spinal cord tumours.

A series of 81 patients operated for intramedullary spinal cord tumours over a span of 12 years (1975-1986) is presented. The mean age of the patients was about 37 years and 9 patients were in the paediatric age (0-15 years). In 29 cases (36%) the tumours were located within the cervical region; in 34 cases the tumours were located in the thoracic region (42%) and in 18 cases the tumours were located within the medullary cone (22%). Ependymoma accounted for 52% of the cases (42 cases), astrocytoma for 20% (16 cases), lipoma for 7% (6 cases), hemangioblastoma for 6% (5 cases), metastatic tumours for 5% (4 cases); in the remaining 8 cases (10%) rare or very rare spinal tumours were found. The 9 patients in the paediatric age and 8 additional cases who had undergone previous surgery were then excluded from this review. In 59% of the patients with ependymoma and in 90% of the patients with astrocytoma severe neurological deficit were already present before the operation. Total tumour removal was accomplished in 81% of the cases with ependymoma, total or subtotal removal was achieved in 50% of the cases with astrocytoma. At long term follow-up study fair or good functional results were observed in 19 out of 41 cases of ependymoma (46%) and in 3 cases out of 10 of astrocytoma (30%). The surgical outcome was mostly related to the preoperative neurological conditions and to tumour malignancy grade.

Intracranial repair of interrupted facial nerve in course of operation for acoustic neurinoma by microsurgical technique.

The microsurgical refinement of the lateral suboccipital approach is, in our opinion, the most satisfactory operative technique for achieving total removal of acoustic neurinomas of all sizes. In this series of 164 operated cases, large or very large tumours accounted for 64% of the cases (105 patients). The facial nerve was sacrificed in about 19% of the cases. In 81% of the cases the facial nerve was respected (65%) or repaired (16%) by direct intracranial suture performed immediately after tumour removal. Good or fair functional results were obtained in about 65% of the cases by this last procedure, which has to be considered as the treatment of choice for facial nerve repair. The results are compared with those of other series and with those obtained by different nerve substitution procedures.

Low doses of dopamine agonists in the long-term treatment of macroprolactinomas.

To evaluate the long-term effects of dopamine agonists in the treatment of macroprolactinoma, we studied prolactin levels and tumor size for 30 to 88 months (57 +/- 14, mean +/- S.D.) in 38 patients treated with bromocriptine or lisuride. Elevated prolactin levels became normal in 30 patients, and the tumor shrank in 29. After two years of treatment, we attempted to reduce the maintenance dose (5 to 20 mg of bromocriptine per day or 0.4 to 0.8 mg of lisuride per day); in 21 patients no changes in prolactin levels or tumor size were observed over 6 to 52 months with 0.625 to 10 mg of bromocriptine per day or 0.05 mg of lisuride per day. However, it was possible to withdraw the drug in only one patient. We conclude that dopamine agonists are usually effective treatments for macroprolactinoma and that after a response has been obtained, it can be maintained in many patients with a greatly reduced dose.

Effect of chronic bromocriptine administration on tumor size in patients with "nonsecreting" pituitary adenomas.

The effect of chronic bromocriptine administration (7.5-20 mg/day for 1-32 months) on the size of "nonsecreting" pituitary adenomas (NPA) was studied in 20 patients. Brain computed tomography showed a marked reduction of the adenoma in one patient after 1 month of treatment (7.5 mg/day); further scans taken 2 and 15 months later, under the same bromocriptine dose, did not show any other variations in the tumoral mass. In the remaining 19 patients, no changes in tumor size were documented by CT during the treatment. Four patients had a worsening of visual fields during bromocriptine administration and they were referred for neurosurgery. In conclusion, bromocriptine was ineffective in reducing tumor size in all but one patient with NPA and, in some cases, it did not prevent tumor growth as is suggested by the worsening of visual fields. Thus, bromocriptine treatment, at least at the doses capable of shrinking macroprolactinomas, seems to be of limited value in patients with NPA.

Medulloblastoma. Results of a sequential combined treatment.

Actuarial progression-free survival rate at 5 years of a series of 34 patients with medulloblastoma treated by combined surgery, radiotherapy, and chemotherapy was 71%. No relapses were observed in 14 patients followed for more than 5 years. Treatment consisted of a short postoperative course of vincristine (VCR) and intrathecal (IT) methotrexate (MTX) followed by irradiation to the entire cranio spinal axis. Maintenance chemotherapy (CCNU, VCR, and IT MTX) was then continued to encompass 2 years from surgery. Failure occurred in nine patients: four had local recurrence, four dissemination within the central nervous system, and one widespread skeletal metastases. Poor prognostic factors such as presence of malignant cells in the cerebrospinal fluid, non-radical surgery, young age, and radiation doses less than 50 Gy to the tumor bed, did not adversely affect the outcome of patients in this series. Long-term sequelae from the treatment program could be observed in all patients, and in 58% they were severe enough to interfere with normal, active life.

Cushing's disease and marked hyperprolactinemia in a patient with a pituitary macroadenoma: effectiveness of bromocriptine treatment.

The case of a young boy bearing a pituitary PRL secreting adenoma (20-30,000 ng/ml) with the unusual association of clinical and endocrinological features of Cushing's disease successfully treated with bromocriptine is described. Brain computed tomography evidenced a huge pituitary adenoma leading to visual field defects and raised intracranial pressure. Due to the very large size of the tumor, which rendered the complete neurosurgical removal unlikely, medical treatment with bromocriptine (10 mg/day) was started. Follow-up for more than six months demonstrated an impressive reduction of tumor size, the lowering of prolactin levels into the normal range, the normalization of visual field, and the regression of both clinical and biochemical signs of hypercortisolism.