Oligoclonal banding in AIDS and hemophilia.

Hypergammaglobulinemia is a consistent finding in patients within the AIDS spectrum and with hemophilia A. Serum samples from patients with these conditions were analyzed for the presence of oligoclonal banding, using a high-resolution serum protein electrophoresis system. The incidence of banding is significantly greater in well homosexuals who are HIV-antibody positive and in patients with pre-AIDS-related complex, AIDS-related complex, AIDS with opportunistic infections, and AIDS with Kaposi's sarcoma than in normal blood donors. The incidence of banding is similar to controls in patients with hemophilia who have received either no blood products, cryoprecipitate only, or limited infusions of factor VIII concentrate. In patients who have received frequent infusions of factor VIII concentrate, the incidence of banding significantly increases. Thirteen of sixty-seven hemophiliac patients developed AIDS or symptoms related to HIV infection independent of their banding pattern. We hypothesize that the bands are not diagnostic of AIDS, but seem to correspond with disease progression, and that they are absent early in the disease, appear later in the course, and may disappear with advanced disease.

Vincristine and prednisone prolong the survival of patients receiving intravenous or oral melphalan for multiple myeloma: Cancer and Leukemia Group B experience.

A total of 589 patients with previously untreated multiple myeloma were randomized to receive daily oral melphalan, pulse-dose intravenous (IV) melphalan, carmustine (BCNU), or lomustine (CCNU). All patients received an initial tapering course of prednisone (Pred). During week 22 (day 154), patients were randomized to receive or not to receive additional therapy with vincristine (VCR) (1 mg/m2) and prednisone (0.6 mg/kg/d for seven days) at 8-week intervals. The influence of VCR/Pred was determined in 302 patients who remained on study beyond 22 weeks after initial therapy. VCR/Pred converted a significant percentage of nonresponders to responders in patients treated with melphalan (55% v 19%, P = .002), but not in patients treated with a nitrosourea (48% v 23%, P = .06). Survival beyond week 22 was significantly longer following the addition of VCR/Pred in patients receiving melphalan (median, 35.3 months v 27.0 months; P = .003) but not in patients receiving BCNU or CCNU (median, 28.1 months v 26.2 months; P = .91). These differences were seen both for oral and IV melphalan. A trend for beneficial effect of VCR/Pred was definitely seen in the good-risk patients (P = .03) but only suggestive for poor-risk patients (P = .12). Following adjustment for VCR/Pred effects, there were no differences in the survival of patients receiving any of the four initial treatments.

Low ionic antiglobulin tests.

Seven serologic procedures were studied to determine their respective value in compatibility and screening tests. All seven were significantly improved by the use of 4 volumes of serum, rather than 1, with 1 volume of red cell suspension, and a low-ionic antiglobin test (LIAGT) was distinctly superior to the other six procedures evaluated. In this test, during the incubation of serum and cells at 37 degrees for 20 minutes, ionic concentration was reduced 62 percent. However, after removal of all supernatant, the red cells were washed three times with an isotonic solution that provided 80 percent reduction in ionic concentration, and the washed cells were tested for their agglutinability with low-ionic (80% ionic reduction) anti-IgG antiglobin reagent. This modified LIAGT was usually more, and apparently never less, sensitive than a test described earlier and is expected to be associated with much less nonspecificity. The extreme sensitivity of LIAGT for many long-term frozen stored alloantiserums is a retained property of the modified test and has been associated with IgG aggregation during storage.

Quantitative cerebrospinal fluid IgG measurements as a marker of disease activity in multiple sclerosis.

The value of various parameters of reporting quantitative cerebrospinal fluid (CSF) IgG levels to indicate disease activity in 34 patients with clinically definite multiple sclerosis was examined. IgG alone correlated significantly with increasing degree of disability and increasing number of clinical central nervous system lesions. There was also a trend toward higher mean IgG levels when the course was relapsing and progressive as opposed to progressive or relapsing. For the IgG index, the relationships were the inverse of that noted with IgG alone. IgG-albumin ratio and IgG synthetic rate did not correlate significantly with course, number of CNS lesions, or degree of disability, and there was no statistically significant relationship between any parameter of reporting quantitative CSF IgG and age, duration of disease, history of recent exacerbation, or area of first involvement in the nervous system. We conclude that although newer methods of reporting CSF IgG elevations in multiple sclerosis are more sensitive and some of them, more specific, in confirming a diagnosis than CSF IgG alone, this parameter remains the best marker of disease activity in individual patients.

Single, sequential, and multiple alkylating agent therapy for multiple myeloma: a CALGB Study.

Four intravenous (IV) alkylating agent regimens were tested in 615 previously untreated patients with multiple myeloma. Patients were randomized to receive melphalan, cyclophosphamide, and carmustine in combination (MCBP), sequentially (Seq-MCBP), or in combination with doxorubicin (MCBPA). The fourth group received IV melphalan (MP) as the only alkylating agent. All groups received a tapering dose of prednisone. Toxicity was similar for all regimens although the nadir of cytopenia was reached more quickly for the regime including melphalan only. Response as measured by reduction in myeloma protein or other parameters were similar for the four treatments. Survival was significantly poorer for the group receiving the alkylating agents in sequence. The survival of high tumor cell load patients who were azotemic was better in the groups treated with IV MP or with the combination of IV MCBP. In view of the simplicity and probable cost savings attached to single-agent treatment, a melphalan/prednisone regimen should be considered as initial therapy for all patients with myeloma.

Isolation and partial characterization of a fatty acid binding protein in rat liver plasma membranes.

When [14C]oleate-bovine serum albumin complexes were incubated in vitro with rat liver plasma membranes (LPM), specific, saturable binding of oleate to the membranes was observed. Maximal heat-sensitive (i.e., specific) binding was 3.2 nmol/mg of membrane protein. Oleate-agarose affinity chromatography of Triton X-100-solubilized LPM was used to isolate a single 40-kDa protein with high affinity for oleate. On gel filtration, the protein comigrated with various fatty acids but not with [14C]bilirubin, [35S]sulfobromophthalein, [14C]taurocholate, [14C]phosphatidylcholine, or [14C]cholesteryloleate. A rabbit antibody to this membrane fatty acid-binding protein gave a single precipitin line with the antigen but no reactivity with concentrated cytosolic proteins, LPM bilirubin/sulfobromophthalein-binding protein, or rat albumin or other rat plasma proteins. The antibody selectively inhibited heat-sensitive binding of [14C]oleate to LPM. Immunofluorescence studies localized the antigen in liver-cell plasma membranes as well as in other major sites of fatty acid transport. These data are compatible with the hypothesis that this protein may act as a receptor in a hepatocellular uptake mechanism for fatty acids.

Quantitative CSF IgG measurements in multiple sclerosis and other neurologic diseases. An update.

To compare four ways of measuring CSF IgG levels in diagnosing multiple sclerosis (MS), we analyzed CSF samples of 106 patients with clinically definite, probable, or possible MS and 127 patients with other diseases. The IgG synthetic rate and IgG index were the most sensitive tests at 0.88 and 0.94, respectively; IgG alone and IgG-albumin ratio, at 0.53 and 0.59, were less valuable. The IgG synthetic rate (0.87) was more specific than the IgG index (0.73), making it the quantitative measure that best correlated with a clinical diagnosis of definite MS. However, combining these four methods showed an even higher correlation. Quantitative CSF IgG elevations occurred much less frequently in patients with clinically definite MS receiving immunosuppressives and in those with clinically probable and possible MS. We did not perform qualitative CSF IgG measurements, but our methods' sensitivity and specificity were comparable with those attributed to oligoclonal IgG bands by others. We also found numerous other diseases where elevations of CSF IgG occurred by all four methods.

Physicochemical and immunohistological studies of a sulfobromophthalein- and bilirubin-binding protein from rat liver plasma membranes.

Affinity chromatography over bilirubinagarose and sulfobromophthalein (BSP)-agarose was used to isolate two proteins, with high affinities for bilirubin and BSP, respectively, from Triton X-100-solubilized rat liver plasma membranes. The protein eluted from either affinity column migrated as a single band of approximately 55,000 D on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and either protein cochromatographed with both [14C]bilirubin and [35S]BSP on Sephadex G-75. On gradient gels without reduction or SDS, or on Sephadex G-150, the native BSP-binding protein had an estimated molecular mass of approximately 100,000 D. After incubation with SDS, an additional Sephadex G-150 peak of molecular mass of 56,000 D was observed. Both, the 100,000- and 56,000-D G-150 peaks cochromatographed with [35S]BSP. The native protein had an isoelectric point of 3.5, stained with periodic acid-Schiff but not Sudan black, and contained 4 mol of sialic acid per mol of protein. A rabbit antibody to the BSP-binding protein gave a line of identity with both the BSP- and bilirubin-binding antigens, and inhibited the binding of [14C]bilirubin and [35S]BSP, but not [14C]oleate or [14C]taurocholate, to rat liver plasma membranes. Immunohistochemical studies revealed the presence of the antigen on all surface domains of rat hepatocytes, but not on other cell populations from normal rat liver. It was not found in other organs. These data are compatible with the hypothesis that a specific liver cell plasma membrane protein mediates the hepatocytic sequestration of bilirubin and BSP.

Studies of oleate binding to rat liver plasma membranes.

The binding of [14C]oleate to rat liver plasma membranes was examined under various conditions in vitro. In protein free incubation mixtures binding was saturable, inhibited by excess cold oleate, virtually abolished by heat denaturation of the membranes, reversible by post-incubation with cold oleate or albumin, and pH and temperature dependent. When [14C]oleate was incubated in the presence of albumin or beta-lactoglobulin, the amount of membrane bound oleate was a function of the calculated free oleate concentration in the incubation mixture. Trypsin significantly inhibited binding. Further analysis suggests that membranes contain 10(15) high affinity (Kav = 2 x 10(8)M-1) oleate binding sites/mg protein.

Comparison of oral melphalan, CCNU, and BCNU with and without vincristine and prednisone in the treatment of multiple myeloma. Cancer and Leukemia Group B experience.

A total of 361 evaluable patients with previously untreated multiple myeloma were randomized to receive oral melphalan (0.15 mg/kg/day for seven days, followed by 0.05 mg/kg/day after recovery from the nadir of the leukocytes), BCNU (150 mg/m2 intravenously every six weeks) or CCNU (100 mg/m2 orally every six weeks). All patients received a tapering six-weeks) or CCNU (100 mg/m2 orally every six weeks). All patients received a tapering six-week course of prednisone starting at 0.8 mg/kg for the first two weeks. At week 22, one-half of the patients were randomized to receive vincristine (1 mg/m2) and prednisone (0.6 mg/kg for seven days) every two months in addition to previous therapy. The melphalan treated patients showed a significantly higher overall objective response frequency (59%), according to Myeloma Task Force criteria, when compared to those treated with BCNU (40%) or CCNU (42%). The survivals for all patients were not statistically different for the three treatment programs. However, the good-risk patients treated with melphalan had significantly longer survival (P = 0.02) than the equivalent patients who received BCNU or CCNU. The addition of vincristine and prednisone at week 2 did not significantly increase the percentage of subsequent objective responses or prolong the subsequent survival of any treatment group. It is concluded that oral melphalan is superior to BCNU and CCNU in producing objective responses and in prolonging survival in good risk patients.

Influence of renal failure on myelosuppressive effects of melphalan: Cancer and Leukemia Group B experience.

The influence of renal insufficiency on melphalan-induced myelosuppression was examined during the initial 10 weeks of treatment in 295 patients with multiple myeloma. Patients were randomized to receive either oral melphalan (0.15 mg/kg/day for 7 days, followed by 0.05 mg/kg/day after recovery from the wbc count nadir) or iv melphalan (16 mg/m2 every 2 weeks for four doses, followed by a single dose every 4 weeks). All patients received a 6-week tapering course of prednisone. Patients with renal insufficiency (BUN greater than or equal to 30 mg/100 ml) had a significantly higher frequency of severe leukopenia (less than or equal to 1000 cells/mm3) following iv melphalan than did patients with normal renal function (50% vs 15%, respectively; P = 0.007). The latter effect resulted in an increased frequency of drug-related deaths secondary to infection. The frequency of severe thrombocytopenia (less than or equal to 25,000 cells/mm3) was also greater in patients with renal failure following iv melphalan therapy. Reduction of iv melphalan dose to 50% in patients with elevated BUN reduced the frequency of these complications to levels that were not significantly different from those observed in patients with normal renal function. The frequency of severe myelosuppression was independent of renal function in patients receiving oral melphalan. Possible explanations for these findings are discussed.

Augmentation of hemagglutination by low ionic conditions.

Short incubation at 37 C, 80 per cent reduction in ionic concentration and removal of liquid phases after each reaction step, provided the basis for the construction of four new serologic tests for alloantibodies to human erythrocytes. In the first, the incubation fluid was replaced with protamine sulfate to aggregate intensely the evaluated red blood cells. After dispersal by phosphate buffer, residual antibody mediated agglutination could be discerned. As a second method, this low ionic polycation (LIP) test was followed by a normal ionic IgG antiglobulin test (LIP-AGT). A third method employed low ionic washing of erythrocytes and low ionic antiglobulin serum (LIAGT). Finally, a modified LIP test was conducted entirely under low ionic conditions and followed by a low ionic antiglobulin test (modified LIP-AGT). LIP, LIP-AGT and LIAGT were successfully employed for all routine blood bank serology tests. Their sensitivity and impact on blood bank performance are described.

Response to therapy in IgG myeloma patients excreting lambda or kappa light chains: CALGB experience.

Sixty-eight previously untreated patients with IgG myeloma who were entered into five protocols of Cancer and Leukemia Group B (CALGB) were studied in order to determine the possible influence of excretion of kappa versus lambda urinary light chains on responses to treatment and survival. All patients in these protocols were included if the serum and urine protein abnormalities were confirmed by one of the two group reference laboratories. Pretreatment characteristics of the two groups of patients did not differ significantly. Of 44 patients with kappa Bence Jones proteinuria, 19 patients (43%) had good responses to treatment, whereas only 3 of 24 patients (13%) with lambda Bence Jones proteinuria had good responses (p = 0.02). Survival for the patients excreting kappa light chains was significantly better than survival for patients excreting lambda chains (median survival 31 versus 12 mo, p = 0.02).

Improved survival of increased-risk myeloma patients on combined triple-alkylating-agent therapy: a study of the CALGB.

Two hundred fifty-two previously untreated evaluable patients with multiple myeloma were entered into a study testing a regimen of three intravenous alkylating agents, melphalan, cyclophosphamide, and carmustine (BCNU), given in combination (BCMP) against a regimen employing oral melphalan (MP). Both regimens included a tapering course of prednisone. Objective responses based on the Myeloma Task Force criteria were significantly more frequent in the group receiving BCMP. Survival for the entire group of BCMP-treated patients was not significantly better than that for MP-treated patients (p = 0.62). However, when the survival of the poor-risk (high tumor cell load) group of patients treated with BCMP was compared with the survival of the poor-risk (high tumor cell load) group of patients treated with MP, an improvement in survival attributable to BCMP therapy was seen (p = 0.049 and 0.02, respectively). In the good-risk (low and intermediate tumor cell load) group, BCMP treatment resulted in a trend toward poorer survival, but this did not achieve statistical significance (p = 0.080 and 0.23, respectively). These results indicate that optimal therapy in myeloma may be dependent on the extent of disease at the time of first treatment. Additional studies to explore the effects of treatment intensity and duration are needed in order to design improved myeloma treatment based on the patient's extent of disease.

Nonuniform distribution of occult blood in feces.

Inhibition of anti-Rh29 by erythrocytic stroma in feces was devised as a specific test for fecal occult blood. The sensitivity of this test was equivalent to that of a standard Hemoccult test, namely, 10(8) erythrocytes/g feces. Comparison of results of this test with results of Hemoccult tests of random stool specimens and of stools following ingestion of autologous blood revealed nonuniform distribution of occult blood in feces. The extent of nonuniformity was determined by testing samples of stool specimens following ingestion of 51Cr-labeled autologous blood. This allowed comparison of Hemoccult, inhibition of anti-Rh, and radioactivity, and showed that the three labels could separate in the feces and that some single small samples of feces could be relatively free of blood while blood was readily demonstrable in other portions. The variability of standard Hemoccult test was somewhat reduced by dispersing the feces in distilled water before performing the test.

Mechanochemical properties of brain clathrin: interactions with actin and alpha-actinin and polymerization into basketlike structures or filaments.

Two molar urea (pH 7.5) and column chromatography on Sepharose 4B were used to separate clathrin (coat protein) from the membrane of coated vesicles from bovine brain. Lytron (polystyrene) particles were used for study of the interaction of clathrin with contractile proteins. Muscle G-actin, F-actin, and alpha-actinin were bound by clathrin-coated Lytron particles, while no interaction was found when muscle tropomyosin and serum albumin were tested. Clathrin molecules dispersed in a solution of 20 mM Tris-HCl (pH 7.5) were found to be elongated. When the pH was adjusted from 7.5 to 6.5, clathrin molecules associated into basketlike or cage structures similar in size and shape to those observed in enriched preparations of coated vesicles. Below pH 6.0, cages or baskets became amorphous aggregates. Raising the pH from 6.5 to 8.0, addition of 5-10 mM ATP or EDTA, or addition of 200 mM KCl resulted in the dissassembly of baskets and the formation of filamentous arrays of various widths. Because of clathrin's biochemical and biophysical properties, its interaction with contractile proteins, and its presence in the membrane of vesicles of various cell types, we classified clathrin in the group of mechanochemical proteins.

alpha-Actinin and tropomyosin interactions with a hybrid complex of erythrocyte-actin and muscle-myosin.

alpha-Actinin isolated from dog muscle was used to incite antibodies in rabbits, Antibodies, purified by affinity chromatography on CNBr-Sepharose coupled with alpha-actinin and then ferritin-labeled were found to localize on the Z disc of muscle sarcomeres. Molecules of alpha-actinin as an adsorbed monolayer on the surface of polystyrene Lytron particles could bind muscle-actin and tropomyosin from solution. Both the ATPase activity and superprecipitation of an erythrocyte-actin and muscle-myosin hybrid actomyosin complex were altered by alpha-actinin, while tropomyosin diminished these alpha-actinin effects. The binding properties of alpha-actinin are consistent with those of an anchoring protein for microfilaments in nonmuscle cells.

Idiotypic specificities of anti-Rh antibodies.

Idiotypic antibodies were produced in rabbits to Rh antibodies isolated from the serum of an individual with high Rh antibody titer. These antisera after absorption with blood IgG failed to react with immunoglobulins lacking anti-Rh activity. They agglutinated cells coated with the immunizing antibody to high titers. Two other anti-Rh coats out of 22 tested also were agglutinated but to considerably reduced titers indicating a degree of anti-Rh cross specificity. Four other antisera made to red cells coated with different anti-Rh antibodies showed definite but weaker idiotypic specificity. Both the idiotypic and cross-idiotypic antigens of the Rh antibodies were completely blocked when univalent fragments of the Rh antibodies were bound to antigen on the red cell.

Pathogenesis of shigella diarrhea. Serum anticytotoxin antibody response produced by toxigenic and nontoxigenic Shigella dysenteriae 1.

The serum antitoxin response to the cytotoxin contained in preparations of Shigella dysenteriae 1 (Shiga's bacillus) exotoxin was studied in natural and experimental infections of man. Natural infection resulted in the rapid appearance of toxin-neutralizing antibody, which disappeared some time between 9 and 18 mo after infection. Experimental infection of human volunteers provided the opportunity to study immunoglobulin class of the antibody in sera obtained serially from 7 to 50 days after infection. Neutralizing antibody was present only in the IgM fraction isolated by sucrose density gradient ultracentrifugation. This was confirmed by the use of solid-phase immunoaffinity chromatography. Even though the time-course and immunoglobulin class of the antitoxin antibody response was similar to that previously observed for anti-O polysaccharide antibody, the biologically active cytotoxin was shown to be highly susceptible to destruction by proteolytic enzymes. Sera from subjects infected with a virulent invasive chlorate-resistant Shiga mutant thought to be "nontoxigenic" also contained antibody which was similarly restricted to the IgM fraction. Biologically active cytotoxin was recovered when this mutant organism was grown in liquid media with controlled ion concentration. The mutant cytotoxin was heat labile, neutralized by antiwild-type cytotoxin antibody, and was separable by isoelectric focusing into two fractions with pI 7.2 and 6.1 like the wild-type toxin. These studies show that cytotoxin antigen is produced during in vivo infection with Shiga bacilli, resulting in a serum antitoxin antibody response. Without explanation is the restriction of the antibody to the IgM class and lack of evidence for an IgG antibody to the protein cytotoxin. Finally, mutant strain 725, previously designated "nontoxigenic," was shown to produce biologically active cytotoxin in vitro and, in experimentally infected volunteers, to result in a serum IgM antibody similar to that observed during infection with the wild-type strain.