A randomized study of antiretroviral management based on plasma genotypic antiretroviral resistance testing in patients failing therapy. CPCRA 046 Study Team for the Terry Beirn Community Programs for Clinical Research on AIDS.

OBJECTIVE: To determine the short-term effects of using genotypic antiretroviral resistance testing (GART) with expert advice in the management of patients failing on a protease inhibitor and two nucleoside reverse transcriptase inhibitors. DESIGN: Prospective randomized controlled trial. SETTING: Multicenter community-based clinical trials network. PATIENTS: One-hundred and fifty-three HIV-infected adults with a threefold or greater rise in plasma HIV-1 RNA on at least 16 weeks of combination antiretroviral therapy. INTERVENTIONS: Randomization was either to a GART group, where genotype interpretation and suggested regimens were provided to clinicians, or to a no-GART group, where treatment choices were made without such input. MAIN OUTCOMES MEASURES: Plasma HIV-1 RNA levels and CD4 cell counts were measured at 4, 8, and 12 weeks following randomization. The primary endpoint was change in HIV-1 RNA levels from baseline to the average of the 4 and 8 week levels. RESULTS: The average baseline CD4 cell count was 230 x 10(6) cells/l and the median HIV-1 RNA was 28,085 copies/ml. At entry, 82 patients were failing on regimens containing indinavir, 51 on nelfinavir, 11 on ritonavir, and nine on saquinavir. HIV-1 RNA, averaged at 4 and 8 weeks, decreased by 1.19 log10 for the 78 GART patients and -0.61 log10 for the 75 no-GART patients (treatment difference: -0.53 log, 95% confidence interval, -0.77 to -0.29; P = 0.00001). Overall, the best virologic responses occurred in patients who received three or more drugs to which their HIV-1 appeared to be susceptible. CONCLUSION: In patients failing triple drug therapy, GART with expert advice was superior to no-GART as measured by short-term viral load responses.

Frequency of antiretroviral drug resistance mutations in HIV-1 strains from patients failing triple drug regimens. The Terry Beirn Community Programs for Clinical Research on AIDS.

The frequency of protease and reverse transcriptase (RT) gene mutations was determined in HIV-1 strains from 153 patients entering the CPCRA 046 (GART) study who were failing triple-drug regimens consisting of one protease inhibitor (PI) and two RT inhibitors. Population-based sequence analyses showed that nearly all patients had similar RT gene mutations regardless of prior drug exposure, although the M184V mutation was significantly less prevalent in patients not recently treated with lamivudine. Whilst typical inhibitor-specific ('signature') protease gene mutations were found in patients failing their first PI, these mutations were significantly less likely to be found in patients exposed to two or more PIs. Protease gene mutations associated with multi-PI resistance were more likely to be observed in patients treated with more than one PI. These results suggest sequential treatment with PIs select for a relatively limited number of protease gene mutations that likely originated during early PI therapy. These protease gene mutations and a similarly limited set of RT gene mutations appear to be responsible for treatment failure in antiretroviral therapy.

High-dose chemotherapy followed by peripheral blood stem cell rescue for metastatic rhabdomyosarcoma: the experience at Chicago Children's Memorial Hospital.

BACKGROUND: Because outcome for metastatic rhabdomyosarcoma remains poor with standard therapy, and because some patients with extensive unresectable metastatic rhabdomyosarcoma are unable to tolerate standard therapy with the associated large radiation fields, peripheral blood stem cell rescue (PBSCR) following high-dose chemotherapy was offered as consolidative therapy for patients with Stage 4/Group IV rhabdomyosarcoma. PATIENTS AND METHODS: Eight patients with Stage 4/Group IV rhabdomyosarcoma were diagnosed from May, 1992, through November, 1994. Consolidative PBSCR following thiotepa 300 mg/M2 on days -7, -6, and -5; cyclophosphamide 1,500 mg/M2 on days -5, -4, -3, and -2; and carboplatin 600 mg/M2 on days -3 and -2 was offered to those patients who achieved a complete remission with multimodality therapy. Patients with extensive metastatic disease who did not receive full doses of radiation to all sites of disease remained eligible for high-dose chemotherapy and PBSCR. RESULTS: Five of eight patients achieved a complete response. Four patients underwent PBSCR. One of the four patients is alive without evidence of disease 53 months post-PBSCR. All other patients died of progressive disease. CONCLUSIONS: These results, along with the existing literature, show no advantage of high-dose chemotherapy followed by PBSCR as consolidative therapy for patients with Stage 4/Group IV rhabdomyosarcoma over standard dose chemotherapy, radiation, and surgery. For patients with extensive, unresectable disease at diagnosis who cannot receive radiation to all areas of disease based on concerns of marrow reserve, high-dose chemotherapy followed by PBSCR does not appear to provide adequate local control and cannot be offered as curative therapy.

Molecular modelling of HLA-DQ suggests a mechanism of resistance in type 1 diabetes.

Genetic resistance and susceptibility to insulin-dependent diabetes mellitus (IDDM) have been associated with the HLA class II region on chromosome 6. In many races, the DQB1*0602 allele has been observed at a decreased frequency in IDDM, suggesting a protective role. A DNA sequence analysis of five patients, previously typed as having allele DQB1*0602, revealed sequence variation: one is DQB1*0603 and four possess unique sequences related to DQB1*0602 (one patient) or DQB1*0603 (three patients). Samples from four unaffected controls possessed normal DQB1*0602 sequences, and all patients and controls have normal DQA1 sequences. Each of the four unique patient sequences yields predicted amino acid sequences differing from the more common DQB1 alleles by variation at codons 9,38 (silent), 59, and/or 62. Molecular modelling of the predicted protein sequence of these permissive variants reveals an HLA-DQ structure from diabetic patients that differs in the surface contour of the peptide-binding groove from normal DQB1*0602 sequence. In all the models of permissive molecules, the surface area corresponding to the HLA-DR pockets 6,7 and 9 are modified. These pockets accommodate side chains of the bound peptide; thus modification of this region could alter peptide specificity. This 'pocket change' suggests that the normal allele could confer dominant protection against the development of IDDM by affecting peptide and/or T cell receptor (TCR) binding. This could regulate the deletion or suppression of T cell clones inappropriately recognizing the beta cells of the pancreas.

Anti-p53 antibodies in sera of workers occupationally exposed to vinyl chloride.

BACKGROUND: The p53 tumor suppressor gene (also known as TP53) is often mutated in a wide variety of cancers, including angiosarcoma of the liver (ASL). Anti-p53 antibodies have been detected in the sera of patients with leukemia, childhood lymphoma, or cancers such as those of the breast, lung, colon, esophagus, and liver (hepatocellular carcinoma). PURPOSE: The objective of this study was to determine the prevalence and time of appearance of serum anti-p53 antibodies during the pathogenesis of ASL associated with occupational exposure to vinyl chloride. METHODS: Enzyme-linked immunoassay (EIA) was used to detect anti-p53 antibodies in 148 serum samples from 92 individuals occupationally exposed (in France or in Kentucky) to vinyl chloride; 15 of these individuals (six from France and nine from Kentucky) had ASL. A subset of coded EIA-positive and EIA-negative sera was further analyzed for anti-p53 antibodies by immunoblotting and immunoprecipitation. Nucleotide sequence analysis of exons 5-8 of the p53 gene was conducted on ASL DNA from six patients. We tested sera from 31 men who had no occupational exposure to vinyl chloride; they made up the control group. Statistical analyses were done using the Kruskal-Wallis chi-squared approximation and the Wilcoxon two-sample test for normal approximation. All P values result from two-sided tests. RESULTS: Fourteen serum samples (from nine individuals) were positive in the EIA. Five of the 15 individuals with ASL were positive for anti-p53 antibodies by EIA, immunoblotting, and immunoprecipitation: one individual at 11.3 and 10.8 years before diagnosis, another at 4 months before and shortly after diagnosis, and three when diagnosed or shortly thereafter. Four of the 77 vinyl chloride-exposed workers without diagnosed ASL were positive for anti-p53 antibodies; two of the four had symptoms related to vinyl chloride toxicity. Tumors from three of the six vinyl chloride-exposed workers from which sufficient DNA for analysis was obtained had A:T to T:A missense mutations of the p53 gene. Anti-p53 antibodies were detected in two of these individuals. Among the control group, two of 15 serum samples from 15 lung cancer patients and zero of 15 serum samples from control subjects without cancer had anti-p53 antibodies as substantially lower levels than the nine (10%) of 92 vinyl chloride-exposed workers who were positive for anti-p53 antibodies. CONCLUSIONS AND IMPLICATIONS: Serum anti-p53 antibodies can predate clinical diagnosis of certain tumors, such as ASL, and may be useful in identifying individuals at high cancer risk, such as workers with occupational exposure to vinyl chloride.

Complement factors H and I synthesized by B cell lines function to generate a growth factor activity from C3.

B lymphocytes and transformed B lymphoblastoid cell lines express CR2 (CD21, C3d/EBV-receptor) that is specific for C3 fragments generated by cleavage of C3b or spontaneously hydrolyzed native C3 (C3i) by the serum enzyme factor I and its cofactor, factor H. It had been shown previously that the Raji B cell line could be cultivated in serum-free medium supplemented with only transferrin and either OKB7 anti-CR2 mAb, C3d, or C3d-derived peptides containing the CR2 binding site. Because these agents appeared to function through ligation of CR2, it was unclear how native C3 could also serve as a growth factor, because C3 does not bind to CR2. It appeared possible that Raji cells might be able to use endogenous factors H and I to generate a CR2 ligand from C3, because previous studies had shown that Raji cells synthesized factor H and probably also synthesized factor I. PCR analysis was used to demonstrate factor I mRNA in Raji cells. Secretion of Raji cell factor I protein was confirmed by a sensitive mAb ELISA. Several B cell lines were examined for C3-dependent growth. Raji cells required both C3 (or OKB7) and transferrin for growth, whereas Wil-2 cells grew with transferrin alone and C3 enhanced the growth-promoting activity of transferrin. Two other B cell lines (Daudi and U698M), the T cell line 8402, and the U937 monocytoid cell line could not be sustained with transferrin plus C3. The C3-dependent growth of Raji cells was inhibited almost completely by either OX-23 anti-factor H or 052.11.3 anti-factor I mAb that also blocked the activity of serum-derived factor H or I, respectively. By contrast, there was no inhibition of growth by either OX-24 anti-factor H or OX-21 anti-factor I mAb that did not block factors H and I activity. After the spontaneous hydrolysis of native C3 to C3i, it is hypothesized that Raji cells convert C3i to iC3i with endogenous factors H and I, and then this iC3i serves as a growth factor by binding to membrane CR2.

Analysis of HLA genotypes and susceptibility to insulin-dependent diabetes mellitus: HLA-DQ alpha complements HLA-DQ beta.

It is well known that certain genes in the HLA-D region confer increased susceptibility to insulin-dependent diabetes mellitus (IDDM). Previous studies have documented an increased risk associated with the HLA-DR beta chain alleles, DR3 and DR4, and the DQ beta chain allele DQB1*0302 (formerly DQw8). Since DQ alpha is also polymorphic and has been strongly implicated as the primary IDDM susceptibility locus in other races, we wanted to assess the contribution of DQ alpha to IDDM in Caucasians. This information would enable us to define more precisely the class II association with IDDM as well as gain insight into issues of cis versus trans association of DQ heterodimers in this disease. To this end, the DQ alpha genotype was determined for a large group of diabetic and normal Caucasian individuals who had been HLA-DQ beta and HLA-DR typed previously. Using the polymerase chain reaction and a set of twelve oligonucleotide probes, we determined the DQ alpha genotype of 323 patients with IDDM and 182 normal subjects. We found that certain DQ alpha alleles are decreased in the diabetic population compared with normal subjects (i.e. DQA1*0102 and *0103), while others are significantly increased in patients with IDDM (i.e. DQA1*0301 and *0501). In addition, certain combinations of DQ alpha alleles are associated with increased susceptibility to disease (i.e. DQA1*0301, *0501). These results parallel our findings at the DQ beta locus; however, because of the various associations between DQ alpha and DQ beta chains, the risks conferred by DQ alpha are generally lower than those at DQ beta. Moreover, our data indicate that, in Caucasians, no single DQ alpha allele accounts for the highest degree of susceptibility to IDDM as in other races, although DQ alpha analysis may be informative in a few cases. When done in combination, however, oligonucleotide analyses at both DQ alpha and DQ beta complement each other and provide a more complete assessment of the HLA-associated component of disease susceptibility in IDDM.

T-cell receptor beta-subunit gene polymorphism and autoimmune disease.

We have investigated the distribution of genotypes of a restriction fragment length polymorphism of the T-cell receptor beta-subunit gene in Caucasoid controls and patients with insulin-dependent diabetes mellitus, celiac disease, dermatitis herpetiformis, and idiopathic membranous nephropathy and also in South Indian controls and diabetics. We found no significant differences between the controls and patients with any disease in either ethnic group, a result which contrasts with previous reports of associations with both insulin-dependent diabetes mellitus and idiopathic membranous nephropathy. However, the most striking finding was a marked disparity between the genotype distribution in our Caucasoid control population and that previously reported by other investigators.

Restriction fragment length polymorphism of the gene encoding the alpha chain of the human T cell receptor.

Two allelic forms of the T cell antigen receptor alpha chain gene were discerned by restriction fragment length polymorphism (RFLP) employing the T cell antigen receptor alpha chain probe pGA5, and the restriction enzyme Bgl II. Analysis revealed that the polymorphic fragments are detected by a probe specific for the constant region exon of the T cell antigen receptor alpha chain gene. Furthermore, the polymorphic fragments were shown to segregate within families. The two allelic forms yield two homozygous states, 3.2/3.2 and 2.9/2.9, at a frequency of 76.5 and 2.9%, respectively, within the normal population. The heterozygous state was observed in 20.6% of the population. The discovery of allelic forms of both the alpha and beta chains of the T cell antigen receptor genes may provide a unique opportunity to study heritable markers of T cell function in several human diseases.

A procedure for the isolation of highly purified populations of B cells, T cells and monocytes from human peripheral and umbilical cord blood.

A sequential separation protocol is described which reproducibly yields biologically active populations of B cells, T cells and monocytes from human peripheral and umbilical cord blood. The purification protocol employed T lymphocyte rosetting with unmodified sheep red blood cells, monocyte adherence to microexudate-coated flasks and anti-immunoglobulin panning of B cells. Population purity was determined by cytofluorographic light scatter analysis, expression of cell surface markers, esterase activity and mitogen responsiveness. The sequential protocol reproducibly yielded viable and functionally active cell populations of greater than 95% purity from a single starting population of mononuclear cells.

Effects of different protein supplements on mitogen responses of human peripheral blood lymphocytes.

We evaluated the effects of 6 supplements often used in human lymphocyte cultures, including fetal calf serum, autologous human serum, pooled human AB serum, hypogammaglobulinemic human serum, bovine serum albumin and human serum albumin. Lymphocyte proliferation of unstimulated and mitogen activated peripheral blood mononuclear cells was measured by [3H]thymidine incorporation. The response of cells stimulated with the T-cell mitogen phytohemagglutinin-P were significantly lower when cultured in bovine serum albumin supplemented media, but were otherwise not supplement dependent. In contrast, responses of cells stimulated with the B-cell mitogens Cowan I strain of S. aureus and antisera against the mu or delta chain of human immunoglobulin were significantly effected by supplement. Cultures containing fetal calf serum and bovine serum albumin had high background responses without a proportional rise in cellular proliferation when B-lymphocyte-specific mitogens were utilized. Autologous human serum and pooled human AB serum contained immunoglobulin which interacted with each of the B cell mitogens, thus limiting their usefulness as in vitro supplements. Cells grown in human serum albumin supplemented media had minimal background and high stimulated responses to B-cell mitogens. These results indicate that human serum albumin is an optimal supplement for in vitro human lymphocyte proliferative assays since it supports high stimulated cell responses with low background activity, is devoid of immunoglobulin and had minimal variability among lots.