Spermatozoa characteristics in six psittacine species using light microscopy.

Even though breeding of companion birds has increased continuously for years, the fecundity assessment of birds has hardly been acknowledged. Knowledge of the structure of spermatozoa is crucial for evaluation of the basic reproductive biology of any species as well as for phylogenetic research and cladistic analyses of internal relationships. Spermatozoa of six different psittacine species (Nymphicus hollandicus, Myiopsitta monachus, Agapornis roseicollis, Melopsittacus undulatus, Tanygnathus lucionensis, Guarouba guarouba) were examined using light microscopy. Head length (nucleus including acrosome), head width, midpiece length and tail length were measured and documented. Significant differences were obvious among almost all of the species for almost all four parameters. However, in all the six species a significant moderate correlation between spermatozoa midpiece lengths and tail lengths (r=0.535, p

Interleukin-2 and interleukin-2 receptor expression in human corticotrophic adenoma and murine pituitary cell cultures.

The production of IL-1 and IL-6 by pituitary cells has recently been demonstrated. In this study we investigated the expression of IL-2 and its receptor (IL-2R) by pituitary cells of different species. In Northern blots, a single hybridizing band of 1 kb, identical to that in normal stimulated lymphocytes, was obtained with specific IL-2 probes. In the mouse AT-20 pituitary tumor cell line, IL-2 mRNA expression was detected after stimulation with corticotropin-releasing hormone or phorbol myristate acetate. In human corticotrophic adenoma cells, basal IL-2 mRNA expression as well as IL-2 secretion were further stimulated by phorbol myristate acetate. Both adenoma and AtT-20 cells showed detectable amounts of IL-2R mRNA and by immunofluorescence, IL-2R membrane expression. In addition, dual immunofluorescence studies in rat anterior pituitary cells demonstrated colocalization of IL-2R with ACTH-positive cells and other cell types expressing the receptor. In addition to the action of lymphocyte-produced IL-2, this cytokine may have a paracrine or autocrine regulatory role within the pituitary. It remains to be established whether IL-2 production occurs in the normal pituitary or is intrinsic to the process of tumor development of these cells. IL-2 may be involved in the growth control of pituitary cells.

Repression of basal transcription by HMG2 is counteracted by TFIIH-associated factors in an ATP-dependent process.

A basal repressor of class II gene transcription was identified, purified, and found to be identical to nonhistone chromosomal protein HMG2. HMG2 was shown to inhibit basal transcription under conditions in which transcription templates form soluble complexes with HMG2. Order-of-addition experiments clearly revealed that HMG2 acted after assembly of a TBP-TFIIA-promoter complex and before formation of the fourth phosphodiester bond by RNA polymerase II. Subsequently, an activity that efficiently counteracted repression of transcription by HMG2 in both TBP- and TFIID-containing transcription systems was isolated. Several lines of evidence suggested that antirepression was mediated by a TFIIH-associated factor. The antirepressor first coeluted with TFIIH, was depleted from this fraction by antibodies directed against the TFIIH subunit p62, was dependent on either ATP or dATP, and then was inhibited by the ATP analogs AMP-PNP and ATP gamma S. Relief of HMG2-mediated repression as well as basal promoter function of TFIIH may involve a helicase that coelutes with TFIIH and displays similar nucleotide specificities. Taken together, these data suggest novel consequences of chromatin-associated HMG proteins and they provide direct evidence for a role of TFIIH-associated enzymes in ATP-dependent antirepression of nonhistone chromosomal proteins.

RNA polymerase II cofactor PC2 facilitates activation of transcription by GAL4-AH in vitro.

We have isolated from a crude Hela cell cofactor fraction (USA) a novel positive cofactor that cooperates with the general transcription machinery to effect efficient stimulation of transcription by GAL4-AH, a derivative of the Saccharomyces cerevisiae regulatory factor GAL4. PC2 was shown to be a 500-kDa protein complex and to be functionally and biochemically distinct from native TFIID and previously identified cofactors. In the presence of native TFIID and other general factors, PC2 was necessary and sufficient for activation by GAL4-AH. Cofactor function was specific for transcriptional activation domains of GAL4-AH. The repressor histone H1 further potentiated but was not required for activation of transcription by GAL4-AH. On the basis of the observation that PC2 exerts entirely positive effects on transcription, we propose a model in which PC2 increases the activity of the preinitiation complex in the presence of an activator, thereby establishing a specific pathway during activation of RNA polymerase II.

A conserved tissue-specific structure at a human T-cell receptor beta-chain core promoter.

The T-cell receptor (TCR) beta-chain promoters have been characterized as nonstructured basal promoters that carry a single conserved ubiquitous cyclic AMP-responsive element. Our investigation of the human TCR beta gene uncovers a surprisingly complex and tissue-specific structure at the TCR Vbeta 8.1 promoter. The core of the promoter (positions -42 to +11) is recognized by the lymphoid cell-specific transcription factors Ets-1, LEF1, and AML1 as well as by CREB/ATF-1, as is demonstrated in gel shift and footprinting experiments. With the exception of LEF1, these factors activate transcription in T cells. Binding sites at the core region show little conservation with consensus sites. Nonetheless, CREB, Ets-1, and AML1 bind and activate cooperatively and very efficiently through the nonconsensus binding sites at the core promoter region. Moderate ubiquitous activation is further induced by CREB/ATF and Sp1 factors through proximal upstream elements. The tissue-specific core promoter structure is apparently conserved in other T-cell-specifically expressed genes such as the CD4 gene. Our observations suggest that both the enhancer and the promoter have a complex tissue-specific structure whose functional interplay potentiates T-cell-specific transcription.

Involvement of negative cofactor NC2 in active repression by zinc finger-homeodomain transcription factor AREB6.

The transcription factor AREB6 contains a homeodomain flanked by two clusters of Krüppel type C2H2 zinc fingers. AREB6 binds to the E-box consensus sequence, CACCTGT, through either the N- or the C-terminal zinc finger cluster. To gain insights into the molecular mechanism by which AREB6 activates and represses gene expression, we analyzed the domain structure of AREB6 in the context of a heterologous DNA-binding domain by transient-transfection assays. The C-terminal region spanning amino acids 1011 to 1124 was identified as a conventional acidic activation domain. The region containing amino acids 754 to 901, which was identified as a repression domain, consists of 40% hydrophobic amino acids displaying no sequence similarities to other known repression domains. This region repressed transcription in vitro in a HeLa nuclear extract but not in reconstituted transcription systems consisting of transcription factor IID (TFIID), TFIIB, TFIIE, TFIIH/F, and RNA polymerase II. The addition of recombinant negative cofactor NC2 (NC2alpha/DRAP1 and NC2beta/Dr1) to the reconstituted transcription system restored the activity of the AREB6 repression domain. We further demonstrated interactions between the AREB6 repression domain and NC2alpha in yeast two-hybrid assay. Our findings suggest a mechanism of transcriptional repression that is mediated by the general cofactor NC2.

Isolation and characterization of an early T-helper/inducer cell line with a unique pattern of surface phenotype, constitutive cytokine secretion and myc oncogene expression.

The cell line AG-F was isolated from the marrow of a neuroblastoma patient undergoing myeloablative treatment and autologous bone marrow rescue. A year later, the patient developed a Hodgkin's type lymphoma. AG-F cell line demonstrated an unusual phenotype, lacking surface CD2 and CD3, but expressing high levels of CD4, CD5, CD7, CD29, and CD45RO. Markers associated with Hodgkin's lymphoma cells, CD15 and CD30, were also positive. AG-F cells grow in suspension in clusters of 50-200 cells, with a doubling time of 9 h. They can also grow in serum-free medium and form tumors in nude mice. AG-F cells have amplified N-myc and c-myc and high levels of the corresponding mRNA transcripts. Cytogenetic analysis revealed a DNA index by flow cytometry of near tetraploid cells and a karyotype of 85-87 chromosomes, with consistent abnormalities in chromosomes 1, 5, and 9. Gene rearrangement studies revealed rearrangement of the beta gene of the T-cell receptor. AG-F cells secrete high levels of IL-6, IL-8, IL-10, and GM-CSF. Cell adherence and formation of long processes could be induced by fibronectin and were enhanced by exposure to PMA. Cells exposed to phorbol myristate acetate (PMA) had increased expression of CD11a, CD11b, CD18, CD45RO, and HLA-DR, whereas expression of CD15 and CD30 was markedly decreased. Similarly, the level of c-myc and N-myc oncoproteins and the levels of the cytoskeletal proteins, actin, tubulin, and vimentin markedly decreased early after PMA-induced differentiation.

Interleukin involvement in anterior pituitary cell growth regulation: effects of IL-2 and IL-6.

The pituitary gland plays a central role in the interactions between the immune and neuroendocrine systems. The expression of receptors for interleukin-1 (IL-1), IL-2, and IL-6 and the intrinsic production of these ILs by pituitary cells have been described. Previous studies have focused on the way cytokines influence hormone secretion. We have determined whether, in addition to these effects, ILs could affect pituitary cell proliferation. In GH3 cells, both IL-2 (1-100 U/ml) and IL-6 (10-500 U/ml) significantly stimulated [3H]thymidine incorporation and cell count. In contrast, inhibitory effects of both IL-2 and IL-6 at the same concentrations were observed on normal rat anterior pituitary cell growth. This finding was clearly evident when cells were cultured in Minimum Essential Medium-D-valine medium, a condition that results in cultures virtually free of fibroblasts. Autoradiographic studies confirmed that [3H]thymidine was only incorporated in the nucleus of nonfibroblastic pituitary cells. No direct correlation between the effects of IL-2 and IL-6 on cell growth and hormone secretion was apparent. By immunofluorescence, we observed IL-2 receptor expression on GH3 cells and, for the normal rat cultures, a high percentage of PRL-secreting and a lower percentage of GH-producing cells expressing IL-2 receptors, providing new evidence for a direct site of action of IL-2 on pituitary cells. Considering that uncontrolled division of cells may result from either excessive growth stimulation or deficient growth inhibition, the regulation of pituitary cell growth by IL-2 and IL-6 together with their intrinsic pituitary production could be of potential importance in pituitary adenoma pathogenesis.

Induction of immature thymocyte proliferation after castration of normal male mice.

The physiological basis and immunological significance of thymic enlargement in castrate male animals is not known. We used normal male C57 Bl/6 mice to examine the contribution of in situ thymocyte proliferation to castration-induced enlargement of the thymus. Animals castrated at 8-10 weeks of age were compared to normal intact males. Thymocytes were examined 4-120 days after castration using flow cytometry to determine DNA content and thus the number of cells in active phases of the cell cycle. These properties were examined in unseparated thymocytes and in phenotypic subpopulations defined by expression of CD3, CD4, and CD8. For thymocytes obtained from intact control glands, a mean of 11.0 +/- 1.0% were in active phases of the cell cycle. The percentage of cycling thymocytes was increased to a mean of 22.5 +/- 1.9% in the week after castration (P < 0.001). This change occurred in the absence of significant thymic enlargement. At 8-10 days after castration, thymic weight increased abruptly to a new steady state which was double that of intact controls (78.0 +/- 4.1 vs. 39.1 +/- 2.6 mg; P < 0.001). In these enlarged glands, only 9.9 +/- 0.8% of cells were cycling, which was not significantly different than controls (P > 0.3). Proliferating cells identified in fixed thymus tissue sections after in vivo administration of bromodeoxyuridine were located in the subcapsular cortex and medulla. Analyses of thymocyte subpopulations indicated that most cycling cells had immature phenotypes (CD4+CD8+, CD4-CD8+, and CD3lo or CD3-). Castrate glands studied in the steady state period 8-120 days after surgery contained significantly fewer CD3+ cells than intact controls (P < or = 0.045). The findings suggest an intrathymic role for androgens in affecting generation of the mature T cell repertoire.

Regulation of oxygen radical release from murine peritoneal macrophages by pharmacologic doses of PGE2.

The ability of pharmacologic doses of PGE2 to alter the release of superoxide (O2-) and hydrogen peroxide (H2O2) from elicited peritoneal macrophages (M theta) was studied. Twice-daily administration of 200 or 100 micrograms of PGE2 to mice during accumulation of peritoneal M theta resulted in a significant reduction in M theta recovery and in the triggered release of H2O2, but not O2-. Cultivation of elicited M theta from normal mice with concentrations of PGE2 in excess of 10(-7) M for 24-48 h resulted in a significant reduction in the triggered release of H2O2, but not O2-. Cultivation for shorter periods of time or with lower concentrations of PGE2 failed to alter H2O2 release. This effect of PGE2 was reproduced by the phosphodiesterase inhibitor theophylline. The ability of PGE2 to inhibit H2O2 release in the presence of normal production of O2- was not prevented by the addition of superoxide dismutase. Cultivation of peritoneal M theta with 10(-5) M PGE2 for 48 h failed to increase intracellular catalase, although increased H2O2 scavenger activity was demonstrated. The inhibition of extracellular release of H2O2, but not O2-, by pharmacologic doses of PGE2 may be one mechanism for the anti-inflammatory action of this compound.

Isolation and characterization of the porcine nuclear factor I (NFI) gene.

This study describes the isolation of a major portion of the gene for nuclear factor I (NFI) including its 5'-flanking region with transcriptional start sites. We screened a porcine liver, genomic DNA library in phage EMBL3A with synthetic oligonucleotides derived from tryptic and cyanogen-bromide peptide sequences obtained from purified NFI protein. The NFI gene is present as a single copy in porcine DNA.

The early posttransplant prognosis of acute renal allograft rejection as determined by detection of cytotoxic antibodies to lymphoid B cell lines.

We have studied serial samples of pretransplant and posttransplant sera for cytotoxic antibodies to lymphoid B cell lines (LCL) in 45 renal allograft recipients. A total of 48 rejection reactions occurred in 31 patients. A comparison of each patient's most reactive posttransplant serum showed a significantly higher reactivity in the ten patients with early allograft failure when compared with the 21 patients with reversible rejections and the 14 patients who had no rejections. Rejection reactions were easily differentiated by comparing the change in cytotoxic reactivity to LCL of recipients' sera drawn at the time of a rejection episode with the reactivity of their pretransplant sera. In 32 rejections considered non-antibody-associated cytotoxic reactivity of recipients' sera to LCL either decreased or remained essentially unchanged during the rejection. In 16 rejections considered antibody-associated the recipients' sera drawn during the rejection episode showed an increase in cytotoxic reactivity ranging from 40% to 100%. Response to antirejection therapy and three month graft survival had a significant correlation with changes in LCL antibody reactivity during a rejection. Only two of the 32 rejections considered non-antibody-associated failed to reverse compared with eight of the 16 antibody-associated rejections (P less than .001). Graft survival at three months in patients with non-antibody-associated rejections was 90% compared with 27% in the 11 patients who had antibody-associated rejections (P less than .001) Other parameters possibly related to the severity of a rejection reaction or to early allograft prognosis did not differ appreciably between the two types of rejections. This included the time posttransplant to the first rejection episode, the number of patients with multiple rejections in the first three months, and rejections requiring dialysis therapy. Determination of a change in cytotoxic reactivity to LCL during a rejection reaction enables one to predict the response to antirejection therapy and early allograft prognosis. This may ultimately be useful in selecting different types of antirejection therapy for individual patients.

Alterations in T lymphocyte subpopulations associated with renal allograft rejection.

The peripheral blood OKT3 (total T), OKT4 (T helper/inducer), and OKT8 (T suppressor/cytotoxic) cells were determined by flow cytometry on twenty consecutive recipients of HLA-nonidentical cadaveric renal allografts. The absolute number of cells in all three populations decreased significantly posttransplantation, but no differences were found between patients experiencing rejection and those in quiescence. An OKT4/OKT8 ratio of greater than or equal to 1.7, either pretransplant or posttransplant, uniformly identified patients who subsequently experienced rejection. However, an OKT4/OKT8 ratio of less than 1.7 did not identify patients with a low risk of rejection. Pretransplant splenectomy was performed in 6 of 7 patients who rejected despite a low ratio. Serial monitoring of the OKT4/OKT8 ratio posttransplantation determined that an increase in the ratio of greater than or equal to 0.5 was a sensitive (81%) and specific (91%) indicator of a rejection episode. Graft survival was improved in patients with a high posttransplant OKT4/OKT8 ratio. These results indicate that the balance of helper and suppressor cell function may be of critical importance to the fate of an allograft, and that the alterations in this balance can be used to assist in the clinical management of allograft recipients.

Monoclonal antibody analysis of responder and stimulator cells in the human autologous mixed lymphocyte reaction.

Responder and stimulator cell subpopulations in the autologous mixed lymphocyte reaction (AMLR) were determined with the OK series of monoclonal antibodies. Mitomycin-C-treated, monocyte-enriched cell populations were used as stimulator cells in the AMLR. Treatment of these monocytes with either OKM and/or OKI monoclonal antibodies and complement resulted in a marked loss of ability of these cells to act as stimulators in the AMLR. Removal of OKT3+ and OKT4+ cells diminished the proliferative responses of AMLR cultures. Interaction of T cells with autologous monocytes resulted in generation of cells capable of suppressing both MLR and AMLR cultures. The suppressor activity of these cells was diminished by treatment with OKI , OKT4 or OKT8 monoclonal antibodies. No cytotoxic activity to autologous or allogeneic monocytes was observed. Additional studies showed an increased number of OKT9 + and OKI + as well as OKT8+ T cells in the AMLR responder cell population. This study indicates that cultures of T lymphocytes with autologous monocytes yield T cell subset(s) which suppress MLR and AMLR reactivity.

Immunophenotyping of acute leukemia by flow cytometric analysis. Use of CD45 and right-angle light scatter to gate on leukemic blasts in three-color analysis.

This article describes a procedure for performing routine three-color flow cytometric analysis for acute leukemia on lysed whole bone marrow preparations. This technique uses the combination of CD45 intensity and right-angle light scatter (RALS) to distinguish leukemic cells from normal lymphocytes, monocytes, neutrophils, eosinophils, and nucleated red blood cells. On this display, leukemic cells occupy a unique blast region characterized by intermediate CD45 density and low RALS, which, in normal marrows, contains less than 5% of the total cells. This approach was applied to 39 cases of acute leukemia and 8 cases of myelodysplasia or myeloproliferative disorders. The estimate of blasts by flow cytometric analysis was correlated highly with morphologic leukemic cell counts over a wide range. Moreover, the pattern seen on the CD45-RALS display was different for different French-American-British subtypes of leukemia, suggesting that this pattern might be useful for categorization. When CD45-peridin chlorophyll alpha protein was combined with other pairs of fluorescein isothiocyanate- and phycoerythrin-conjugated reagents, it was possible to set an analysis window on the leukemic blasts and display dual-parameter (ie, green vs. red fluorescence) data regarding expression of two additional markers on the leukemic population. This gating strategy was superior to traditional forward-angle versus RALS displays in that it did a better job of isolating the leukemic cells analytically.

Phenotype of the hairy cells of leukemic reticuloendotheliosis defined by monoclonal antibodies.

Hairy cell populations of greater than 90 purity were prepared from six samples obtained from four patients with leukemic reticuloendotheliosis (LRE). These then were analyzed for surface immunoglobulin (SIg) and antigens specified by a panel of monoclonal antibodies. The hairy cells from all patients displayed SIg and antigens reactive with OKIa-1 and OKM-1 antibodies; these markers often were expressed simultaneously. T-cell-specific antigens were not displayed on the surface of hairy cells. The simultaneous expression of SIg and OKM-1 which ordinarily are unique for B cells or myeloid cells, respectively, suggests that hairy cells may represent an aberrant form of either cell type with defective regulation of antigen expression. It alternatively suggests the possibility that B cells and myeloid elements develop along a common pathway and that the hairy cell is a component of such a pathway.

Reimbursement for clinical flow cytometry testing.

Throughout the United States, reimbursement for new laboratory technology is not well understood by the clinicians and laboratory personnel who provide it. It is important to understand how new technology will be paid for in today's changing, more tightly controlled, health-care funding environment. For flow cytometry testing, specifically, this confusion is not just restricted to providers of the tests. The carriers from many states still list flow cytometry as "investigational", contrary to the 1988 HCFA ruling. Although the HCFA ruling is not subject to interpretation by state Medicare carriers, it is often up to the provider whose claims are being denied to educate the carrier concerning its mistake. When confronted with the appropriate evidence (sometimes including a phone call from the regional HCFA office), most carriers relent. It is critical to note that claims must be billed correctly. This means that approved ICD-9-CM codes must be used with the appropriate CPT-4 codes. If there is no prevailing rate established in the state or if it has been established prohibitively low, it will be necessary to lobby and educate the carrier in regard to the medical necessity of the testing, as well as justifying the cost. This can be done by providing the medical director of the carrier with all necessary patient information, laboratory reports, and scientific literature validating the use of the technology for each individual claim. It may also require submitting cost justification data supporting the request. The importance of the development of a good working relationship with one's local Medicare carrier cannot be overemphasized. This relationship is a critical component of establishing a fair and appropriate rate for the reimbursement of any test.

Subacute cutaneous lupus erythematosus in multiple members of a family with C2 deficiency.

Deficiency of the second component of complement (C2d) has been associated with systemic lupus erythematosus (LE)-like syndromes as well as recurrent infections. In particular, C2d has been associated with the LE subset of subacute cutaneous LE (SCLE), the presence of anti-Ro antibodies (anti-Ro or SS-A), and the human leukocyte antigen (HLA) types A25, B18, and DR2. A family with C2d in which three members have developed SCLE was observed and studied clinically, serologically, and immunogenetically. Deficiency of the second component of complement was present in all six family members, while anti-Ro was present in only two. There was a strong but incomplete association of C2d and SCLE with HLA-DR2, but the association was not complete with positivity of anti-Ro or antinuclear antibodies. Study of this family reconfirmed the close association of HLA-A25, -B18 and -DR2 with the C2 gene, but indicated a less close association of these loci with serologic markers.

Contributions of flow cytometry to the analysis of the myelodysplastic syndrome.

As the population of North America ages, the incidence of MDS is likely to rise. Epidemiologic survey instruments need to be put in place to document changes in the incidence. The basic mechanism of disease in MDS is largely unknown. No unifying, testable hypothesis is yet available, but apoptosis, with mitochondria playing a key role, are central to any discussion of MDS. Cytogenetic abnormalities have not provided an explanation of MDS but are of diagnostic and prognostic significance. The emergence of immunologic factors is of major importance and emphasizes the need for early detection. Flow cytometry can be used diagnostically to exclude other causes of cytopenias, document the phenotypic manifestations of myeloid dysmaturation, and provide blast enumeration. The distinctions between MDS and acute leukemia are arbitrary, and the process should be conceptualized as a continuum. There is a need for continued work to establish minimal diagnostic criteria for MDS. The current prognostic scoring systems do not incorporate findings from the newer technologies.

Mechanism of prostaglandin E2 inhibition of acute changes in vascular permeability.

In order to determine the mechanism of antiinflammatory activity, prostaglandin E2 (PGE2) or diluent was administered to rats 2 h prior to intradermal injections of various mediators of inflammatory vascular permeability changes. Vascular permeability was measured as the accumulation of [125I]rat serum albumin at the site of mediator injunction. PGE2 at 500 micrograms significantly inhibited protein leakage produced by histamine, platelet activating factor, zymosan, and zymosan-activated plasma. Pretreatment with PGE2 had no effect on protein leakage induced by injection of lysosomal enzymes, glucose oxidase, or xanthine oxidase. The accumulation of polymorphonuclear leukocytes (PMNs) at the site of injection of zymosan or zymosan-activated plasma was not altered by PGE2 administration. In separate experiments, the ability of PGE2 to alter phagocytosis and oxygen radical production by PMN was examined. PGE2 significantly inhibited phagocytosis at 2 h, but this returned to normal by 6 h. Production of hydrogen peroxide by PMN was not affected by PGE2. These results suggest that PGE2 prevents acute changes in vascular protein leakage by preventing endothelial cell contraction and by inhibiting specific PMN functions.