Reduced-intensity conditioning using fludarabine, melphalan and thiotepa for adult patients undergoing haploidentical SCT.

Haploidentical SCT (HaploSCT) has been most commonly performed using a myeloablative, TBI-based preparative regimen; however, the toxicity with this approach remains very high. We studied the feasibility of a reduced-intensity conditioning regimen in a phase II clinical trial using fludarabine, melphalan and thiotepa and antithymocyte globulin (ATG) for patients with advanced hematological malignancies undergoing T-cell depleted HaploSCT. Twenty-eight patients were entered in the study. Engraftment with donor-derived hematopoiesis was achieved in 78% of patients after a median of 13 days. Six patients experienced primary graft failure, three out of four tested patients had donor-specific anti-HLA antibodies (DSA) (P=0.001). Toxicity included mostly infections. A total of 21 out of 22 patients with AML/myelodysplastic syndrome (MDS) achieved remission after transplant (16 with relapsed/refractory AML). Five out of the 12 patients (42%) with AML/MDS with <15% BM blasts survived long term as compared with none with more advanced disease (P=0.03). HaploSCT with this fludarabine, melphalan and thiotepa and ATG RIC is an effective, well-tolerated conditioning regimen for patients with AML/MDS with low disease burden at the time of transplant and allowed a high rate of engraftment in patients without DSA. Patients with overt relapse fared poorly and require novel treatment strategies.

HLA class I diversity in Kolla Amerindians.

Human leukocyte antigen (HLA) class I polymorphism was studied within a population of 70 unrelated Kolla Amerindians from the far northwest of Argentina close to the Bolivian border. The results indicate that the HLA-A, -B, and -C alleles typical of other Amerindian populations also predominate in the Kolla. These alleles belong to the following allele groups: HLA-A*02, *68, *31, *24, HLA-B*35, *15, *51, *39, *40, *48, and Cw*01, *03, *04, *07, *08, and *15. For the HLA-A locus, heterogeneity was seen for HLA-A*02 with A*0201, *0211, and *0222; and for A*68 with *68012 and *6817, the latter being a novel allele identified in this population. Analysis of HLA-B identified heterogeneity for all Amerindian allele groups except HLA-B*48, including the identification of the novel B*5113 allele. For HLA-C heterogeneity was identified within the Cw*07, *04, and *08 groups with Cw*0701/06, *0702, *04011, *0404, *0803, and *0809 identified. The most frequent "probable" haplotype found in this population was B*3505-Cw*04011. This study supports previous studies, which demonstrate increased diversity at HLA-B compared with HLA-A and -C. The polymorphism identified within the Kolla HLA-A, -B, and -C alleles supports the hypothesis that HLA evolution is subject to positive selection for diversity within the peptide binding site.

Interleukin 2 induction of proliferation in resting T lymphocytes requires contact with monocytes.

Resting human T cells are known to express significant numbers of intermediate but none or barely detectable low and high affinity IL-2 receptors (IL-2R). IL-2 alone failed to induce proliferation in these cells. However, in presence of small proportion of autologous monocytes, as low as 22 pM, IL-2 induced high levels of proliferation in resting T cells. Introduction of a semi permeable membrane between the two cell types or addition of an anti-CD11b mAb inhibited such induction of proliferation by IL-2. Neither recombinant IL-1 nor IL-1-containing cell-free extracts from activated monocytes substituted for intact monocytes. Autologous B cells failed to replace monocytes. Using antigen-specific cloned human T cells we have shown a lack of requirement for antigen. The proliferation was inhibited by anti-IL-2R alpha mAb. IL-2 appears to be unique since neither IL-4 nor IL-6, alone or in presence of monocytes, led to induction of proliferation in resting T cells. Combination of IL-2 and monocytes induced proliferation in all T cell subpopulations (CD4+, CD8+, CD45RA+ and CD45RO+) and antigen-specific clones examined. It also induces mRNA and surface expression of IL-2R alpha, appearance of high affinity IL-2R and induction of proliferation in large proportions of T cells. As in humans, the IL-2 induction of proliferation in murine resting T cells required contact with syngeneic monocytes, suggesting that such a mechanism of T cells activation is highly conserved.

HLA-A*6817, identified in the Kolla Amerindians of North-West Argentina possesses a novel nucleotide substitution.

Analysis of HLA polymorphism of the indigenous populations of Central and South America has identified many alleles not seen previously in other populations. We have described a novel allele, B*5113, previously in a Kolla Amerindian individual from North-West Argentina. Here we present a second novel allele from this population: A*6817, which differs from its closest neighbour A*68012 by a single substitution at nucleotide 419. This substitution of adenosine 419 A*68012 for thymidine in A*6817 results in a novel amino acid change (aspartate to valine) at residue 116.

A novel HLA allele, HLA-B*5113, identified in the Kolla Amerindians of North-West Argentina.

A novel HLA-B51 allele, B*5113, was identified in a Kolla Amerindian individual from North-West Argentina. HLA-B*5113 differs from B*51011 by two nucleotide substitutions, one synonymous, the other nonsynonymous. The resulting amino acid difference at residue 116 in the HLA-B molecule's peptide binding site is likely to affect the nature of the peptides which bind to this molecule. The finding of this novel allele supports previous findings of increased diversity at HLA-B in Amerindian groups.

Rheumatoid arthritis and its association with HLA-DR antigens. II. Antibodies to native connective tissue antigens detected by enzyme linked immunosorbent assay.

The distribution of frequencies of HLA-DR alloantigens in HLA-DR4 negative subjects was determined in patients with Rheumatoid arthritis (RA) and normal individuals. An increased incidence of HLA-DR1 alloantigen in DR4 negative RA patients (45.9%) compared with DR4 negative healthy controls (23.6%) was found. The difference became significant when the incidence of DR1 was compared between patients with severe disease stages (III-IV) (75%) in contrast to 32% of incidence in patients of the milder stages (I-II) (p less than 0.05). Using Enzyme Linked Immunosorbent Assay we have determined the incidence of serum antibodies to native bovine type I and type II collagens and proteoglycans in patients with RA. Presence of serum antibodies to native type I collagen was detected in 59% of patients with RA, 60% of sera exhibited reactivity to type II collagen and 12% had antibodies to proteoglycans. There was no correlation between the presence of antibodies to type I and II collagens and disease stages, however, the incidence of serum antibodies to proteoglycans was increased in severe disease stages. On the other hand, the presence of high levels of antibodies to type I collagen was associated to HLA-DR1 antigen, (p less than 0.05).

Comparison of tanned cell hemagglutination and counter immunoelectrophoresis test in the detection of antibodies to extractable nuclear antigens (ENA) and to DNA in the systemic lupus erythematosus.

A comparative study of tanned cell hemagglutination (TCH) and counterimmunoelectrophoresis (CIE), two easy and reliable methodes for the routine detection of antibodies against nuclear antigens was performed. Antibodies against ENA, RNA-ase sensitive ENA and DNA antigens were searched in patients affected by systemic lupus erythematosus (SLE). Analysis of the results obtained for a particular antibody using TCH and CIE techniques suggests that both methods should be used for the detection of antibodies to nuclear antigens since in several cases antibodies were detected by one method and not for the other. Besides, the frequency of antibodies to ENA, RNA-ase sensitive ENA and DNA revealed by both techniques is similar to the results reported by others employing laborious tests. TCH and CIE serve as a screen to determine the presence of an antibody system and seems to provide the sensitivity enough as to be used in the smaller routine laboratories that wish to provide services for antibodies to nuclear antigens. When tanned cell hemagglutination was used looking for antibodies to DNA or ENA in the sera of patients affected by SLE it proved to be useful since in samples where antibodies to DNA were absent or at very low titres, antibodies to ENA were present in titres ranging from 1:9 to 1:6 561. The authors think this is of considerable importance since the variety of clinical features seen in different subjects with SLE is often accompanied by different specificities or types and amounts of autoantibodies and that certain combinations of these antibodies coincide with specific clinicopathological abnormalities.

Rheumatoid arthritis and its association with HLA-DR antigens. I. Cell mediated immune response against connective tissue antigens.

HLA-DR antigens and cellular sensitivity to native bovine type I and type II collagen and proteoglycans were examined in patients with classic rheumatoid arthritis (RA) and normal individuals. Fifty eight percent of patients with RA (n = 88) and 28% of normals (n = 52) were DR4+ (pc less than 0.01). DR4 phenotype was significantly increased in patients with severe disease stages (III-IV), as defined by the ARA criteria, in contrast to those showing mild disease stages (I-II) (p less than 0.05). Furthermore, peripheral blood mononuclear cells from 55 patients and 30 controls were evaluated for the in vitro production of leukocyte inhibitory factor in response to native type I and type II collagen and proteoglycans. By using this assay, cells from the arthritic group exhibited a statistically significant response when stimulated with native type I collagen and proteoglycans. The cellular immune response was not associated with any particular HLA-DR antigens, or to the disease stage or severity.

Humoral autoimmune response to rat male accessory glands. Comparative specificity studies of auto and heteroantibodies.

A comparison of specific antibodies induced by a saline extract of chemically modified rat male accessory glands (MRAG) in isologous (male and female rats) and heterologous animals (rabbits and a goat) was made. This study was focused on the specificity of the antibodies confronted with saline extract of rat male accessory glands (RAG), their Sephadex G-100 fractions and autoantigen fragments. Specificity studies with Sephadex G-100 fractions of RAG showed that whereas in male rats only the antibodies reactive with Fraction 1 containing autoantigens were detected, one additional population of antibody with different specificity was revealed in female sera. In contrast, antibodies against several other macromolecules were present in heterologous sera. When analyzed for the serum specificity against three enzymatic fragments of autoantigen, the main specificities of male and female sera were different.

Characterization of an antigen from rat male accessory glands.

An isolated soluble antigen involved in the autoimmune response against rat male accessory glands migrated as a double band when it was submitted to analytical disc polyacrylamide gel electrophoresis and as a homogeneous molecular species in SDS electrophoresis. The purified antigen had a molecular weight of approximately 78 K by SDS electrophoresis and 80 K by gel filtration chromatography. The antigen was identified as a protein and displays androgen dependency. The purified fraction was pathogenically active in microgram doses and induced humoral and delayed-type hypersensitivity responses. Besides, lesions were observed in the target organ. The cellular infiltration was located in prostate glands according to the localization of antigen by immunofluorescence findings.

Regulation of experimental allergic encephalomyelitis. Part 5. Role of the recipient in suppressor cell induction.

Suppressor cells that regulate experimental allergic encephalomyelitis (EAE) are present in spleens of Lewis rats that have recovered from the disease, as demonstrated by adoptive transfer of suppression to normal recipients. However, lethally irradiated recipients (850 rad) of spleen cells from recovered donors are not protected against EAE. Indeed, onset of EAE is accelerated in these irradiated recipients. These findings suggest that the host participates in the suppression of EAE.

Humoral autoimmune response to rat male accessory glands. Effect of castration on the humoral response.

The ability to induce antibodies to rat male accessory glands in male and female rats was demonstrated, but a higher response with wider specificity was revealed in female animals. In order to investigate whether this different response may be influenced by sexual hormones we castrated male and female rats at 4 or 30 days after birth. After that we studied the course and specificity of their humoral response to male accessory glands comparing them with that of sham-operated, sex-matched, littermate controls. Orchidectomy in male or oophorectomy in female rats changed neither the course nor specificity of humoral immune response. We conclude, therefore, that hormonal factors do not play any important role in the experimental model under study.

Isolation of rat male accessory glands autoantigens by affinity chromatography.

The autoantigens of rat male accessory glands were isolated by a short procedure which involved 1) immunization of rats with chemically modified rat male accessory glands' saline extract; 2) purification of immunoglobulin G (IgG) from the autoantisera by chromatography on DEAE-Sephadex A-50;3) coupling of rat IgG anti-rat male accessory glands with 4-B activated Sepharose; 4) addition of rat male accessory glands' saline extract and removal of autoantigens by glycine-HCl, pH 2.9. The purity of the eluted autoantigens was determined by polyacrylamide disc gel electrophoresis (PAGE). These components retained their immunologic activity as demonstrated by inhibition of tanned cell hemagglutination and double immunodiffusion gel precipitation.

Induction of humoral autoimmune response to rat male accessory glands.

Ratas macho y hembra de la cepa Wistar fueron isoinmunizadas con extracto de glandulas accesorias masculinas quimicamente modificadas o sin tratamiento siguiendo diferentes esquemas.La mayoria de los animales produjeron autoanticuerpos convencionales y homocitotropicos, especificos de organo y especie. Los anticuerpos hemaglutinantes fueron 2-ME resistentes en todos los casos. La inmunizacion con el material modificado permitio detectar mayor frecuencia de respuesta y titulo de anticuerpos mas elevados (p < 0,05) sin cambiar la especificidad. La modificacion quimica del material antigenico cumple un papel adyuvant