Axolotl MHC class II beta chain: predominance of one allele and alternative splicing of the beta1 domain.

The axolotl MHC is composed of multiple polymorphic class I loci linked to class II B loci. In this report, evidence of the existence of one class II B locus (Amme-DAB) that codes for two different transcripts is given. A 2.1-kb transcript is translated to a complete beta chain and a shorter transcript of 1.8 kb encodes a molecule lacking the beta1 domain. For two complete class II B mRNA synthesized, up to one mRNA devoid of the beta1 domain is synthesized. Alternative splicing involving a peptide binding domain at a class II B locus evidenced in axolotl (Ambystoma mexicanum) is also observed for A. tigrinum, the tiger salamander. Very little variability is found among various axolotl MHC class II B cDNA sequences, and the same allele is obtained from inbred and wild axolotls. The transcription of one MHC class B locus in two class II B isoforms in thymic cells and in splenic lymphocytes may shed new light on the well-known deficient immune responder state of the axolotl.

Natural and induced apoptosis during lymphocyte development in the axolotl.

Lymphocytes apoptosis was characterized in a urodele amphibian, the axolotl, by morphology using electron microscopy and by flow cytometry after propidium iodide staining, as well as by biochemical criteria with the detection of DNA ladders after glucocorticoid treatment. The morphological and biochemical features observed in treated axolotls are in accordance with the criteria of apoptosis found in different models of mammalian lymphocyte programmed cell death. The onset of natural apoptosis was then detected by DNA fragmentation in thymus and in spleen during lymphocyte development and ontogenesis. A typical DNA ladder characteristic of apoptosis is detectable in the thymus as early as 5 months; apoptosis increases and peaks at 8 months, and is no longer detected by 10 months or thereafter. The ability of a superantigen, Staphylococcus aureus enterotoxin B (SEB), to induce T lymphocyte apoptosis in larvae was investigated as well. In vivo exposure of young axolotl larvae to SEB induces, as in mammals, thymocyte apoptosis as indicated by the enhancement of DNA fragmentation. These last results, natural programmed cell death and SEB induced apoptosis during thymic ontogeny, are discussed in correlation with what is known during mammalian thymic selection and apoptosis.

Structure of MHC class I and class II cDNAs and possible immunodeficiency linked to class II expression in the Mexican axolotl.

Despite the fact that the axolotl (Ambystoma spp. a urodele amphibian) displays a large T-cell repertoire and a reasonable B-cell repertoire, its humoral immune response is slow (60 days), non-anamnestic, with a unique IgM class. The cytotoxic immune response is slow as well (21 days) with poor mixed lymphocyte reaction stimulation. Therefore, this amphibian can be considered as immunodeficient. The reason for this subdued immune response could be an altered antigenic presentation by major histocompatibility complex (MHC) molecules. This article summarizes our work on axolotl MHC genes. Class I genes have been characterized and the cDNA sequences show a good conservation of non-polymorphic peptide binding positions of the alpha chain as well as a high diversity of the variable amino acids positions, suggesting that axolotl class I molecules can present numerous antigenic epitopes. Moreover, class I genes are ubiquitously transcribed at the time of hatching. These class I genes also present an important polylocism and belong to the same linkage group as the class II B gene; they can be reasonably considered as classical class Ia genes. However, only one class II B gene has been characterized so far by Southern blot analysis. As in higher vertebrates, this gene is transcribed in lymphoid organs when they start to be functional. The sequence analysis shows that the peptide binding region of this class II beta chain is relatively well conserved, but most of all does not present any variability in the beta 1 domain in inbred as well as in wild axolotls, presuming a limited antigenic presentation of few antigenic epitopes. The immunodeficiency of the axolotl could then be explained by an altered class II presentation of antigenic peptides, putting into question the existence of cellular co-operation in this lower vertebrate. It will be interesting to analyze the situation in other urodele species and to determine whether our observations in axolotl represent a normal feature in urodele amphibians. But already two different models in amphibians, Xenopus and axolotl, must be considered in our search for understanding immune system and MHC evolution.

Differential response to a stress stimulus of proenkephalin peptide content in immune cells of naive and chronically stressed rats.

Proenkephalin peptides produced by endocrine and nervous tissues are involved in stress-induced immunosuppression. However, the role of peptides produced by immune cells remains unknown. The present study examines the effect of acute and chronic foot-shock stress on proenkephalin peptide content in bone marrow (BMMC), thymus (TMC), and spleen (SMC) rat mononuclear cells. Proenkephalin was not processed to met-enkephalin in BMMC, while in TMC and SMC met-enkephalin represented 10% and 26% of total met-enkephalin-containing peptides, respectively. Naive rats receiving a stress stimulus showed a significant decrease of proenkephalin derived peptides in BMMC, TMC and SMC. However, in chronically stressed rats that already showed basal low peptide levels, a new stress stimulus produced a differential response in each immune tissue. That is, in BMMC peptide levels reached control rats values; in TMC remained unmodified; and in SMC, although precursors content increased, met-enkephalin levels were even lower than those observed in acutely stressed rats. Free synenkephalin content paralleled met-enkephalin changes in SMC of acutely and chronically stressed rats. The in vitro release of met-enkephalin and free synenkephalin increased in SMC of stressed rats. Met-enkephalin produced in SMC and partially processed proenkephalin peptides detected in BMMC, were only found in macrophages. However, met-enkephalin only appeared in bone marrow macrophages after at least 4 h of cell culture. Altogether, these results suggest that a stress stimulus induced proenkephalin peptide release from immune tissue macrophages. The differential response observed in chronically stressed rats suggest an alternative activation of heterogeneous proenkephalin-storing macrophage subpopulations.

Intranuclear network of 3-5 nm and 8-10 nm fibers in EL-4 lymphoma cells.

While much evidence indicates a high degree of spatial organization in the nucleus, the underlying molecular structures that support it remain poorly characterized. By extracting with high concentrations of RNase A in a modification of the sequential extraction protocol of Penman, we have identified a novel intranuclear network in the mouse lymphoma cell line, EL-4. Micrographs of embedment-free sections of extracted cells reveal anastomosing filaments of two different diameters: 3-5 nm and 8-10 nm. The 3-5-nm filaments are interconnected in many junctions and appear to blend smoothly into each other. The 8-10-nm fibers frequently split into two 3-5-nm filaments. Some 3-5-nm fibers appear to be connected at 90 degrees angles with the 8-10-nm fibers. All junctions are smooth with no apparent junction protein. Flow cytometric analysis of RNase A- (and DNase I-) extracted nuclear matrices indicates that they do not contain significant amounts of protein that react with anti-actin and anti-vimentin monoclonal antibodies. Extraction of EL-4 nuclear matrices with high salt does not reveal 8-10-nm core filaments described after similar treatment of tumor cell lines of cervical and mammary origin. The novel characteristics of the core filaments in EL-4 lymphoma cells may reflect cell-type specificity of the nuclear matrix.

Antibodies against the amino-terminal portion of pro-enkephalin inhibit DNA synthesis in human peripheral mononuclear cells.

Pro-enkephalin (PENK) mRNA and PENK-derived peptides have been reported in lymphocytes, monocytes, and macrophages. Met-enkephalin (ME) and/or synenkephalin (SYN)-containing peptides are produced and released by human peripheral blood lymphocytes (HPBL) activated with phytohemagglutinin (PHA). Furthermore, SYN (PENK 1-70) was cleaved to low-molecular-mass peptides in HPBL. In this work we studied the effect of a mouse monoclonal antibody (mAb) and a rabbit antiserum (pAb) against the C-terminal portion of SYN on DNA synthesis in PHA-activated HPBL. [3H]Thymidine incorporation into HPBL incubated with 0.1 microgram/ml of PHA was tested in the presence of different concentrations of mAb immunoglobulin (Ig) G or different dilutions of pAb. mAb induced a concentration-dependent decrease of [3H]thymidine incorporation into HPBL: 7%, 19%, 28%, and 35% of inhibition was observed with 0.1, 1, 1.5, and 2 micrograms IgG, respectively, reaching values of 65% with 10 micrograms IgG. Similarly, pAb dilutions of 1/500, 1/1000, 1/2000 and 1/4000 inhibited DNA synthesis by 63%, 61%, 43%, and 30%, respectively. The inhibitory effect of mAb and pAb was specific since it was not produced by non-immune mouse IgG or several non-immune rabbit sera and was completely reversed by 1 microM of the synthetic peptide [Tyr63](syn 63-70) synenkephalin. These results suggest that low-molecular-mass SYN-derived peptides released by PHA-activated HPBL may participate in the proliferative response of these cells. This is further evidence that the non-opioid portion of PENK--that is, SYN-derived peptides--may be involved in tissue development.

Expression of ICAM-1 (CD54) on normal and leukaemic B cells: implication for the mixed lymphocyte reaction.

Peripheral blood normal B lymphocytes were found to be poor stimulators in the mixed lymphocyte reaction (MLR), in contrast to normal activated B cells which were strong stimulators. This increased capacity to stimulate a strong MLR correlated with an increased expression of the ICAM-1 (CD54) molecule on the surface of these cells. Similarly, the capacity of leukaemic B cells to induce an allogenic stimulation in the MLR was limited to the ICAM-1 (CD54) positive leukaemic cells. The ability of normal activated or leukaemic B cells to induce an MLR was blocked by antibodies directed against ICAM-1.

[T lymphoma of immature phenotype associated with polycythemia vera]. / Linfoma T de fenotipo inmaduro asociado a una policitemia vera.

A 20-year old patient is presented with generalized lymphadenopathy, splenomegaly, hyperleukocytosis and a bone marrow biopsy showing panmyelosis with predominance of immature granulocytes. Lymph node biopsy showed a histopathological feature that was diagnosed as a chronic granulocytic leukemia in blast crisis. The cell surface phenotype of these blast cells showed predominance of immature CD1+, CD7+ T lymphocytes. The T cell lineage was confirmed by DNA rearrangement studies. In addition, the patient showed erythrocytosis, arterial O2 saturation of 92% and thrombocytosis, characteristics of polycythemia vera. After chemotherapy, the patient relapsed with similar symptoms and lymph node cells of similar immature T phenotype. With a revised diagnosis of immature T cell lymphoma associated to a myeloproliferative disorder and polyglobulia, the patient received a combined treatment of Cyclophosphamide-Adriamycin-Vincristine-VM26-Prednisone. Two months later, the patient relapsed again. He received the first phase of induction of the BFM protocol, with partial clinical remission. Five months later, the patient returned with fever, polyadenopathy and splenomegaly. Lymph node cells showed again immature T cell phenotype. The patient was next treated with the m-BACOD scheme, with no response and progression of the disease and he died few days later due to massive bleeding and cardiorespiratory failure.

Linfoma T de fenotipo inmaduro asociado a una policitemia vera / T lymphoma of immature phenotype associated with polycythenia vera

Se presenta un paciente de 20 años con poliadenopatías, esplenomegalia hiperleucocitosis y una biopsia de médula ósea que mostró una panmielosis con predominio d eelementos inmaduros. El estudio histopatológico de la biopsia de un ganglio linfático sugirió el diagnóstico de leucemia mieloide crónica en crisis blástica. El fenotipo inmunológico de las células blásticas mostró predominio de celulas T con fenotipo inmaduro CS1+, CD7+. El linaje celular T se confirmó por estudios de reordenamiento genético. Presenta además eritrocitosis, saturación arterial de O2 de 92% y trombocitosis, características de policitemia vera. Luego de quimioterapia Vincristina y Prednisona, recae a los dos meses con síntomas similares y con células de ganglio linfático del mismo fenotipo T inmaduro. Se replantea el diagnóstico como linfoma T asociado a un síndrome mieloproliferativo y policitemia, y se lo trata con Ciclofosfamida-Vincristina-VM26-Prednisona. Luego de una segunda recaída, dos meses después, se le indica un protocolo BFM, al que responde parcialmente. Cinco meses después el paciente presenta una tercera recaída, donde las células de ganglio muestran nuevamente fenotipo T inmaduro. No responde a un tratamiento con esquema m-BACOD, la enfermedad progresa, falleciendo luego de una hemorragia masiva por un paro cardiorespiratorio

Linfoma T de fenotipo inmaduro asociado a una policitemia vera / T lymphoma of immature phenotype associated with polycythenia vera

Se presenta un paciente de 20 años con poliadenopatías, esplenomegalia hiperleucocitosis y una biopsia de médula ósea que mostró una panmielosis con predominio d eelementos inmaduros. El estudio histopatológico de la biopsia de un ganglio linfático sugirió el diagnóstico de leucemia mieloide crónica en crisis blástica. El fenotipo inmunológico de las células blásticas mostró predominio de celulas T con fenotipo inmaduro CS1+, CD7+. El linaje celular T se confirmó por estudios de reordenamiento genético. Presenta además eritrocitosis, saturación arterial de O2 de 92% y trombocitosis, características de policitemia vera. Luego de quimioterapia Vincristina y Prednisona, recae a los dos meses con síntomas similares y con células de ganglio linfático del mismo fenotipo T inmaduro. Se replantea el diagnóstico como linfoma T asociado a un síndrome mieloproliferativo y policitemia, y se lo trata con Ciclofosfamida-Vincristina-VM26-Prednisona. Luego de una segunda recaída, dos meses después, se le indica un protocolo BFM, al que responde parcialmente. Cinco meses después el paciente presenta una tercera recaída, donde las células de ganglio muestran nuevamente fenotipo T inmaduro. No responde a un tratamiento con esquema m-BACOD, la enfermedad progresa, falleciendo luego de una hemorragia masiva por un paro cardiorespiratorio (AU)

Linfoma T de fenotipo inmaduro asociado a una policitemia vera. / [T lymphoma of immature phenotype associated with polycythemia vera]

A 20-year old patient is presented with generalized lymphadenopathy, splenomegaly, hyperleukocytosis and a bone marrow biopsy showing panmyelosis with predominance of immature granulocytes. Lymph node biopsy showed a histopathological feature that was diagnosed as a chronic granulocytic leukemia in blast crisis. The cell surface phenotype of these blast cells showed predominance of immature CD1+, CD7+ T lymphocytes. The T cell lineage was confirmed by DNA rearrangement studies. In addition, the patient showed erythrocytosis, arterial O2 saturation of 92

and thrombocytosis, characteristics of polycythemia vera. After chemotherapy, the patient relapsed with similar symptoms and lymph node cells of similar immature T phenotype. With a revised diagnosis of immature T cell lymphoma associated to a myeloproliferative disorder and polyglobulia, the patient received a combined treatment of Cyclophosphamide-Adriamycin-Vincristine-VM26-Prednisone. Two months later, the patient relapsed again. He received the first phase of induction of the BFM protocol, with partial clinical remission. Five months later, the patient returned with fever, polyadenopathy and splenomegaly. Lymph node cells showed again immature T cell phenotype. The patient was next treated with the m-BACOD scheme, with no response and progression of the disease and he died few days later due to massive bleeding and cardiorespiratory failure.

Proenkephalin system in human polymorphonuclear cells. Production and release of a novel 1.0-kD peptide derived from synenkephalin.

In the hematopoietic system a pluripotent stem cell generates precursors for lymphoid and myeloid lineages. Proenkephalin-derived peptides were previously detected in differentiated lymphoid cells. We have studied whether the proenkephalin system is expressed in a typical differentiated cell of the myeloid lineage, the neutrophil. Human peripheral polymorphonuclear cells contain and release proenkephalin-derived peptides. The opioid portion of proenkephalin (met-enkephalin-containing peptides) was incompletely processed, resulting in the absence of low molecular weight products. The nonopioid synenkephalin (proenkephalin 1-70) molecule was completely processed to a 1.0-kD peptide derived from the COOH-terminal. This molecule was characterized in neutrophils by biochemical and immunocytochemical methods. The chemotactic peptide FMLP and the calcium ionophore A23187 induced the release of the proenkephalin-derived peptides, and this effect was potentiated by cytochalasin B. The materials secreted were similar to those present in the cell, although in the supernatant a higher proportion corresponded to more processed products. The 1.0-kD peptide was detected in human, bovine, and rat neutrophils, but the chromatographic pattern of synenkephalin-derived peptides suggests a differential posttranslational processing among species. These findings demonstrate the existence of the proenkephalin system in human neutrophils and the production and release of a novel 1.0-kD peptide derived from the synenkephalin molecule. The presence of opioid peptides in neutrophils suggests their participation in the inflammatory process, including a local analgesic effect.

Differential expression of CD25 and HC2 antigens on subtypes of acute myeloid leukemias.

The presence of CD25 and HC2 antigens in 66 different patients with acute myeloid leukemia (AML) was investigated. The expression of both antigens was observed in 32% of AML cells. Dual fluorescence staining experiments performed in 5 AML patient cells showed that CD25 and HC2 antigens were simultaneously expressed in a single cell. The expression of both antigens was mainly observed in the M4 and M5 subtypes of AML. Although immunopurified IL-2 was able to block the binding of anti-CD25 monoclonal antibody to the AML cells, the IL-2 receptor did not appear to be functional.

Mitogenic activation of the human lymphocytes induce the release of proenkephalin derived peptides.

Several reports indicate that enkephalins participate in lymphocyte proliferation and several events of the immune response. It has been proposed that peptides involved in these processes may originate in the nervous system or endocrine glands. We have found that human peripheral blood lymphocytes (PBL) activated with a mitogenic agent contain and release proenkephalin derived peptides. The kinetics of met-enkephalin and cryptic products of proenkephalin in PBL activated with phytohemaglutinin (PHA) were studied. Peptides were released to the supernatant of stimulated PBL, reaching the highest values after 18 to 24 hours. The material secreted corresponds to high, intermediate and low molecular weight peptides derived from proenkephalin, displaying met-enkephalin and synenkephalin (proenkephalin 1-70) immunoreactivity. Therefore, an intrinsic lymphocytic proenkephalin system is induced by PHA and may play an important role in the regulation of the immune response.