Impaired blastogenic response of lymphocytes from synovial fluid and peripheral blood of patients with rheumatoid arthritis

Synovial fluid lymphocytes from patients with rheumatoid arthritis demonstrated a markedly diminished blastogenic response to both phytohemagglutinin and pokeweed mitogens, when compared to normal peripheral blood lymphocytes. The blastogenic response to rheumatoid peripheral blood lymphocytes to both mitogens was also depressed, when compared to the response of normal lymphocytes, but the difference was less marked and was within limits which could be accounted for by recent salicylate therapy. Lymphocytes of both peripheral blood and synovial fluid of rheumatoid patients showed a delayed response to PHA (five days to achieve maximum thymidine incorporation vs four days for normals).     

The activating effect of synovial fluid and washings of synovial membrane on autologous lymphocytes in rheumatoid arthritis

The effect of synovial fluid and washings of synovial membrane on autologous lymphocytes from patients with rheumatoid arthritis and other diseases has been studied using a rapid method based upon the increase in intranuclear birefringence occurring in the early stages of lymphocyte activation. Retardation of polarized light indicating increased lymphocyte activation was seen in lymphocytes from patients with rheumatoid arthritis but not in lymphocytes from patients with other diseases.     

Complement and immunoglobulins in synovial fluid from synovectomized patients with rheumatoid arthritis

In order to see if the complement (C) consumption and conversion, which are typical of rheumatoid joints, continue after synovectomy, 23 knee joints in which synovectomy had been performed from 4-5 to 6-5 years previously, were studied. The mean ratio of the concentration of C3, C4, and C5 in synovial fluid to that in palsma of the same patient was significantly lower than the corresponding ratio for the total protein content. This was found both in joints with active arthritis and in joints without clinical signs of arthritis, and in both seropositive and seronegative patients. Conversion products of C3 were found in 7 of the synovial fluids. The study thus indicated that the complement alterations in synovectomized joints are very similar to those in nonsynovectomized rheumatoid joints. In one synovial fluid agarose electrophoresis showed multiple sharp bands in the gamma region. By crossed immunoelectrophoresis some bands seemed to contain IgG with one type of light chains only. In plasma of the same patients the bands were much weaker, indicating local production of oligoclonal IgG in the joint.     

Anti-Fc gamma receptor III autoantibody is associated with soluble receptor in rheumatoid arthritis serum and synovial fluid

We sought to detect anti-Fc gamma receptor (Fc gamma R) autoantibodies and soluble Fc gamma R in the serum and synovial fluid (SF) from patients with rheumatoid arthritis (RA) and to correlate these serological abnormalities to the polymorphonuclear cell (PMN) activation state. Paired samples of blood and SF were obtained from 33 RA patients as well as blood from 25 normal adults from SF from 20 non-RA patients. Anti-Fc gamma R autoantibodies were assessed by an enzyme-linked immunosorbent assay (ELISA) using recombinant human Fc gamma R as the substrate. Soluble Fc gamma RIII was determined by an ELISA based on the combination of two monoclonal antibodies (MAb). The mean fluorescence intensity (MFI) of complement receptor 1 (CD35) and 3 (CD11b) and Fc gamma RIII (CD16) was evaluated by flow cytometry on the membrane of PMN. IgM anti-Fc gamma RIII activity was present in seven RA sera and five SF, and IgG in eight RA sera and six SF. The average concentration of soluble Fc gamma RIII was 1.80 +/- 3.50 micrograms/ml in RA patients and 0.33 +/- 0.06 micrograms/ml in normal adults (P < 0.05). This was elevated in the SF of 15 RA, while normal in that of all the non-RA patients. There was an inverse correlation between the CD16 MFI on the PMN and the serum/SF soluble Fc gamma RIII level, whereas the density of CD35 and CD11b was markedly augmented. Anti-Fc gamma RIII activity exists in RA patients, associated with soluble Fc gamma RIII. PMN activation could be due to these autoantibodies and thereby obviate the clearance of immune complexes.     

Levels of circulating intercellular adhesion molecule 1 and E-selectin in serum and synovial fluid of juvenile chronic arthritis patients

OBJECTIVE: To measure the levels of two adhesion molecules (AM), soluble intercellular adhesion molecule 1 (sICAM-1) and soluble E-selectin (sE-selectin), in serum and synovial fluid (SF) of patients with juvenile chronic arthritis (JCA). METHODS: Both soluble AM levels were tested, in serum and synovial fluid (SF) samples, with an enzyme-linked immunosorbent assay (ELISA) method. RESULTS: Serum levels of sICAM-1 and sE-selectin in JCA patients were not significantly different from those of a control group. Synovial fluid levels of sICAM-1, but not of sE-selectin, assayed significantly higher (p < 0.05) in JCA patients than in controls. Moreover SF levels of both molecules correlated negatively with disease duration, being higher in the earliest phases. No significant correlations were found between JCA sICAM-1 and sE-selectin levels and leukocyte count or ESR. CONCLUSIONS: These observations may signify a more important role of ICAM-1 than E-selectin in the migration of inflammatory cells into JCA SF. The negative correlation of both AMSF levels in JCA patients with disease duration could reflect a higher expression of ICAM-1 and E-selectin during the earliest phases of the disease.     

Immunological findings in serum and synovial fluid in patients with rheumatoid arthritis (author's transl)

Sera and synovial fluid were investigated in 45 patients with rheumatoid Arthritis and 50 patients with osteoarthritis in inflammatory exacerbation (control group). The following tests were performed: IgG, IgM, IgA determinations, complement components C3, C3, C4, C3-proactivator, ceruloplasmin, electrophoresis, LDH and total acid phosphatase. 1. Serum levels of the ceruloplasmin, alpha 1, alpha 2 and gamma fractions of electrophoresis are significantly higher in patients with rheumatoid arthritis than in patients with osteoarthritis. 2. Synovial fluid: a) There is a significantly higher concentration of IgG, IgM, IgA, C3-proactivator and total acid phosphatase in the synovial fluid of patients with rheumatoid arthritis. b) C4 is significantly lower in patients with rheumatoid arthritis. c) Both groups were also compared with the help of a point system. Every patient received a plus point when the following criteria were seen: IgM greater than 150 mg/100 ml, C3 greater than 50 mg/100 ml, ceruloplasmin greater than 35 mg/100 ml, alpha 1 greater than 0.21 g%, alpha 2 greater than 0.44 g%, beta greater than 0.60 g% and gamma fraction on electrophoresis greater than 0.90 g%. Another point was added if the criteria ceruloplasmin greater than 22 mg/100 ml and C4 less than 17 mg/100 ml were simultaneously seen. With the help of this points system 48 out of the 50 osteoarthritis patients (96%) received zero points, one received 1 point and one 2 points, as opposed to the patients with rheumatoid arthritis where 35 out of 45 (78%) received one or more points. d) The differentation is not improved through additional testing of the rheumatic factors.     

Multiple T cell expansions are found in the blood and synovial fluid of patients with reactive arthritis

OBJECTIVE: To look for evidence of T lymphocyte expansions in the blood and synovial fluid (SF) of patients with reactive arthritis (ReA). METHODS: Paired peripheral blood and synovial samples from 10 patients with ReA were studied by dual color flow cytometry using T cell receptor (TCR) V beta specific and CD4 or CD8 specific antibodies. Two synovial CD8 expansions were studied by 3 color flow cytometry. Peripheral blood samples from 13 healthy, age matched individuals were studied as controls. RESULTS: Statistically significant expansions were observed in all patients, occurring in blood and SF CD4 and CD8 compartments, but were most common in the synovial CD8 compartment. Expansions studied in further detail displayed an activated "memory" phenotype. A synovial BV22S1/CD8 expansion was seen in 5/6 patients with sexually acquired ReA. CONCLUSION: Multiple T lymphocyte expansions are found in both the blood and SF of patients with ReA. Expansions were most commonly found in the synovial CD8 compartment, where they appeared to express both activation and memory markers. This indicates that T lymphocytes (and in particular cytotoxic T cells) may play a pathogenic role in ReA. These findings are consistent with either an antigen or a superantigen driven response.     

T helper 1 response is dominant and localized to the synovial fluid in patients with Lyme arthritis

Cytokines produced by subsets of CD4+ T helper cells responding to an infection influences the efficiency with which the host is able to mount a protective immune response. In an attempt to elucidate the population of active cells involved in the propagation of Lyme arthritis we have utilized intracellular cytokine staining to analyze the polyclonal immune response at the single cell level. We have determined the Th phenotype in the synovial fluid of patients with a variety of chronic inflammatory arthritides, including patients representative of the spectrum of Lyme arthritis. Th1 cells dominate the immune response in the synovial fluid of patients with Lyme as well as those with rheumatoid or other types of chronic inflammatory arthritis. In addition, the severity of Lyme arthritis directly correlates with the ratio of Th1 to Th2 cells in the synovial fluid, such that the larger the effusion, the higher the ratio (r = 0.67, p < 0.05). These results suggest that Th1 cells play a direct role in the pathogenesis of the inflammatory process seen in Lyme arthritis, and that Th2 cells modulate the pro-inflammatory response generated by Th1 cells in the joint. Finally, we identify Th1 cells specific for outer surface protein A of Borrelia burgdorferi, the agent of Lyme disease. These cells are restricted to patients with Lyme arthritis and are localized to the joint. Furthermore, they persist in patients with prolonged antibiotic treatment-resistant Lyme arthritis, suggesting the possibility of an autoimmune process.     

Use of the isolator 1.5 microbial tube for culture of synovial fluid from patients with septic arthritis

Synovial fluid specimens obtained from patients with arthritis were plated onto solid media (conventional cultures) or inoculated into an Isolator 1.5 microbial tube (Isolator cultures), and the yield and time to detection of organisms were compared. Overall, 144 specimens obtained from 137 patients were processed, and 31 (21.5%) cultures obtained from 29 patients were positive by at least one method. Staphylococcus aureus was isolated from 12 patients, Streptococcus pneumoniae and Kingella kingae were isolated from 4 patients each, group G streptococci were isolated from 3 patients, Staphylococcus epidermidis and members of the family Enterobacteriaceae were isolated from 2 patients each, and Streptococcus mitis and Peptostreptococcus prevotii were isolated from 1 patient each. Overall, the causative organism was detected in 31 of 31 (100.0%) Isolator cultures and 24 of 31 (77.4%) conventional cultures (P < 0.02). Twenty-nine of 31 (93.5%) positive Isolator cultures and 20 of 24 (83.3%) conventional cultures were positive by the second day of incubation. Among the 24 cultures positive by both methods, higher numbers of CFU per milliliter were detected with the Isolator system in 13 cultures and with conventional cultures in 2 cultures (P < 0.002). Inoculation of synovial fluid into an Isolator 1.5 microbial tube improves the recovery of organisms causing septic arthritis.     

Biased T-cell antigen receptor repertoire in Lyme arthritis

A common concern with many autoimmune diseases of unknown etiology is the extent to which tissue T-lymphocyte infiltrates, versus a nonspecific infiltrate, reflect a response to the causative agent. Lyme arthritis can histologically resemble rheumatoid synovitis, particularly the prominent infiltration by T lymphocytes. This has raised speculation about whether Lyme synovitis represents an ongoing response to the causative spirochete, Borrelia burgdorferi, or rather a self-perpetuating autoimmune reaction. In an effort to answer this question, the present study examined the repertoire of infiltrating T cells in synovial fluid from nine Lyme arthritis patients, before and after stimulation with B. burgdorferi. Using a highly sensitive and consistent quantitative PCR technique, a comparison of the T-cell antigen receptor (TCR) beta-chain variable (Vbeta) repertoires of the peripheral blood and synovial fluid showed a statistically significant increase in expression of Vbeta2 and Vbeta6 in the latter. This is remarkably similar to our previous findings in studies of rheumatoid arthritis and to other reports on psoriatic skin lesions. However, stimulation of synovial fluid T cells with B. burgdorferi provoked active proliferation but not a statistically significant increase in expression of any TCR Vbeta, including Vbeta2 and Vbeta6. Collectively, the findings suggest that the skewing of the TCR repertoire of fresh synovial fluid in Lyme arthritis may represent more a synovium-tropic or nonspecific inflammatory response, similar to that occurring in rheumatoid arthritis or psoriasis, rather than a specific Borrelia reaction.     

Selective accumulation of related CD4+ T cell clones in the synovial fluid of patients with rheumatoid arthritis

The role of T cells in the pathogenesis of rheumatoid arthritis (RA), especially in the perpetuation of advanced disease, remains unclear. Previous studies have focused on the TCR repertoire of synovial T cells in an attempt to determine whether the pattern of expression is characteristic of Ag-stimulated populations. However, the results of past studies have been conflicting. In the present work, we have undertaken an extensive analysis of the TCRs expressed by CD4+ T cells freshly isolated from synovial fluid of different joints and blood in three patients with established RA. Despite marked heterogeneity of synovial TCR expression, the results showed that 20 to 30% of the TCR beta-chain gene (TCRB) sequences found in one joint were also expressed in a second joint, but not in peripheral blood T cells of the same individual. Analysis of expressed TCRB complementarity-determining region 3 sequences showed the presence of multiple expanded clonal populations that were not predicted by quantitation of beta-chain variable region (Vbeta) expression by immunofluorescence staining. These studies also demonstrated sets of related, but different, complementarity-determining region 3 nucleotide sequences that encoded identical or highly homologous beta-chain amino acid sequences. Analysis of matching T cell clones derived from the joint by limiting dilution culture confirmed coexpression of highly homologous TCR alpha-chain gene (TCRA) and TCRB sequences. Together, these studies suggest that a significant proportion of synovial CD4+ T cells has been selected and expanded by conventional Ag(s) in this disease.     

Coexpression of CD69 and HLADR activation markers on synovial fluid T lymphocytes of patients affected by rheumatoid arthritis: a three-colour cytometric analysis

Haploid and diploid strains of Aspergillus nidulans have been repeatedly treated with the strong mutagen 6-N-hydroxylaminopurine (HAP) which causes only base substitutions. An enormous amount of variability may be rapidly accumulated in haploid or diploid strains of A. nidulans. In particular, in the diploids the analysis of the results shows that after 12 cycles of treatment the conidia differ from each other for about ten recessive lethals and therefore probably for several hundreds of mutations. The viability of the heterozygous multiply mutant diploids is not appreciably different from that of untreated controls. In the diploid strains the accumulated variability was very high. The treatment of a haploid strain during vegetative growth also caused a strong accumulation of mutations, even though deleterious, because they can be maintained in the heterokaryotic condition.     

Functional analysis of rheumatoid factor-producing B cells from the synovial fluid of rheumatoid arthritis patients

OBJECTIVE: To understand the regulation of rheumatoid factor (RF) production in rheumatoid arthritis (RA), we studied IgM-RF production by B cells isolated from the synovial fluid (SF). METHODS: Highly purified SF and peripheral blood (PB) B cells were isolated by negative selection in a fluorescence-activated cell sorter (FACS) and then cultured with either L cells, CD40 ligand (CD40L)-transfected L cells, or type B synoviocytes in the presence or absence of interleukin-2 (IL-2), IL-4, or IL-10. Total IgM and IgM-RF were detected after 14 days by enzyme-linked immunosorbent assay. Enzyme-linked immunospot assays were performed to detect cells that spontaneously produced immunoglobulin. SF B cells were also phenotypically characterized by FACS analysis. RESULTS: Terminally differentiated CD20-,CD38+ synovial plasma cells (PC) present in the SF of RA patients secreted IgM-RF in the absence of a stimulus. IgM-RF production markedly increased when SF B cells were cultured in the presence of type B RA synoviocytes together with IL-10, but independently of CD40-CD40L interaction. Although CD20-,CD38+ PC could also be demonstrated in SF B cells from patients with other forms of arthritis, IgM-RF production was restricted to the SF B cell cultures of patients with seropositive RA. The frequency of IgM-RF-producing cells among IgM-producing PC in patients with seropositive RA was estimated to be as much as 50%. CONCLUSION: These data demonstrate that terminally differentiated CD20-,CD38+ IgM-RF-producing B cells are specifically present in the inflamed joints of patients with seropositive RA. There is evidence that the local environment in the rheumatoid joint favors RF production. The relatively high frequency of IgM-RF PC in the SF B cell population provides evidence of a dominant RA-specific antigen-driven response in the development of the synovial PC repertoire.     

Analysis of type 1 and type 2 T cells in synovial fluid and peripheral blood of patients with rheumatoid arthritis

OBJECTIVE: It has been reported that CD4+ helper T cells play an important role in the pathogenesis of rheumatoid arthritis (RA). We evaluated the presence of intracellular cytokines interleukin 4 (IL-4) and interferon-gamma (IFN-gamma) produced by CD4+ and CD8+ T cells in the synovial fluid and peripheral blood of patients with RA at the single cell level. METHODS: We used 3 color flow cytometric analysis. Synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) were stimulated with phorbol myristate acetate (PMA) and calcium ionophore. The stimulated SFMC and PBMC were triple stained with conjugated mononuclear antibodies (Mab) against cytokines and surface antigens after fixation and permeabilization with a saponine buffer solution. The cells were analyzed for intracellular cytokines (IFN-gamma, IL-4) and surface antigens (CD3, CD4, CD8) using a flow cytometer. RESULTS: The CD4/CD8 ratio was significantly lower in SFMC than in PBMC. The positive rates of IFN-gamma producing cells among CD4+ T cells were significantly higher than those of IL-4 producing cells in both the SFMC and the PBMC of patients with active RA. In the SF of these patients, we also found CD8+ T cells that produce IL-4 alone, or both IL-4 and IFN-gamma. CONCLUSION: In the SF of patients with RA, CD4+ type 1 T cells, which may infiltrate into the synovium and cause pathogenic immune responses in the tissue, are predominant. We believe this cell type also induces migration and activation of CD8+ type 2 T cells into the active site of inflammation, which appears to downregulate the activity of CD4+ type 1 T cells, modulating the excess immune response.     

Induction of invasive and degradative phenotype in normal synovial fibroblasts exposed to synovial fluid from patients with juvenile rheumatoid arthritis: role of mononuclear cell population

OBJECTIVE: To investigate the effect of synovial fluid (SF) from patients with juvenile rheumatoid arthritis (JRA) on proliferation and induction of degradative and invasive phenotype in normal synovial fibroblasts, and to elucidate the contribution of SF cells to this activity. METHODS: SF and/or conditioned medium (CM) from SF cells were evaluated for their ability to (1) stimulate a proliferative response, (2) induce the "activated phenotype" capable of invading cartilage matrix, and (3) promote the release of key matrix metalloproteinases (MMP) in normal synovial fibroblasts. RESULTS: Proliferation of normal synovial fibroblasts exposed to SF or CM from SF cells of patients with JRA was up to 3 times greater than untreated controls. Concomitant with induction of an activated phenotype in the treated synovial fibroblasts, the activated form exhibited up to 250% invasiveness of cartilage matrix compared to untreated synovial fibroblasts (100%), in addition to releasing increased MMP activity, not normally associated with these quiescent cells. This induction was not solely due to tumor necrosis factor-alpha, transforming growth factor-beta, interleukin 1beta (IL-1beta), and IL-6, as SF and/or CM depleted of these cytokines sustained about 40% of their invasive and inducing ability. We observed that the mononuclear cell (MNC) population that infiltrated into the joint cavity secretes this "inducing activity," which can be maintained in culture up to several weeks. CONCLUSION: Our data suggest that the cellular component of SF releases soluble factor(s) that directly or indirectly contribute to (a) proliferation of synovial fibroblasts, and (b) production and release of extracellular MMP by synovial fibroblasts, thereby inducing a degradative and invasive phenotype culminating in cartilage and bone destruction.     

T-cell receptor peptide vaccination in the treatment of rheumatoid arthritis

In several human T-cell-mediated autoimmune diseases and animal models of such illnesses, T-cell receptors (TCR) specific for antigens that initiate or perpetuate the disease share a limited number of variable region determinants. Vaccinations with peptides derived from over-represented TCRs are effective treatment for some of these disorders. RA is a chronic inflammatory disease in which there is prominent T-cell infiltration in the synovial lining layer. TCR V beta 3, V beta 14, and V beta 17 have been found to be over-represented among IL-2 receptor-positive T-cells from patients with RA. A phase II clinical trial in RA, using a combination of three peptides derived from V beta 3, V beta 14, and V beta 17, has yielded promising results. Larger clinical efficacy and safety studies must be performed to determine if TCR peptide vaccination will become a viable treatment alternative for patients with RA.     

T-cell receptor repertoire in matched MART-1 peptide-stimulated peripheral blood lymphocytes and tumor-infiltrating lymphocytes

Characterization of tumor-associated antigens (TAAs) recognized by CTLs makes the consideration of therapeutic strategies based on peptide stimulation of peripheral blood lymphocytes (PBLs) feasible. Several such approaches are adoptive transfer of peptide-stimulated PBLs, ex vivo peptide stimulation of dendritic cells, and direct vaccination with TAA-derived peptides. A critical component of any of these peptide-based strategies is the requirement that the patient's PBLs are able to react productively against the presented TAA. The purpose of this study, through the study of T-cell receptor (TCR) usage, was to evaluate the T-cell response in matched MART-1(27-35) peptide-stimulated PBLs and tumor-infiltrating lymphocytes (TILs). MART-1(27-35)-reactive PBL and TIL cultures were generated from three patients by in vitro stimulation with an immunodominant peptide of MART-1 (MART-1(27-35)). All cultures had a human leukocyte antigen A2-restricted, MART-1(27-35)-specific CTL response. The TCR usage of each was assessed by the DNA sequence analysis of 50 TCR beta clones obtained by rapid amplification of cDNA ends per culture. TCR analysis suggests a TCR repertoire that differed from patient to patient (8-16 subfamilies were used) and a predominant usage of a different variable beta chain (BV) by each of these MART-reactive T cells. These predominant BV rearrangements were derived from multiple clonotypes because different variable, diversity, and junctional regions were observed. However, a similar pattern of expansion was present for both PBLs and TILs; the relative usage of each prevailing BV was more marked in TILs (36, 50, and 78% of TILs versus 26, 20, and 24% of PBLs, respectively), a broader TCR repertoire was used by PBLs (P > 0.05), and similar TCR subfamily usage was noted when TIL and PBL cultures from the same patient were compared (8 of 11, 7 of 9, and 7 of 8 for patients 1, 2, and 3, respectively). Furthermore, the exact same clonotypes derived from predominant TCR subfamilies in the PBLs and TILs were present in each patient, suggesting peptide-stimulated expansion in both biological compartments. These studies suggest that there will not be a limited and predictable TCR subfamily response to a specific TAA, although reproducible patterns of PBL and TIL expansion are present from patient to patient. Additionally, identical T-cell clonotypes having the same potential for antigen-driven expansion were present in a patient's PBLs and TILs. As such, our data support the conceptualization of approaches using adoptive transfer or vaccination based on TAA-derived peptide stimulation of PBLs.     

Ferritin subunits in sera and synovial fluids from patients with rheumatoid arthritis

OBJECTIVE: To determine the proportion of glycosylated ferritin [ferritin bound to concanavalin A (Con-A)] and ferritin subunits in sera and synovial fluids (SF) from patients with rheumatoid arthritis (RA). METHODS: Ferritin concentrations were measured by a sandwich ELISA using rabbit IgG F(ab')2 anti-human ferritin antibody as a coating antibody. Proportions of glycosylated ferritin were examined using Con-A Sepharose 4B. Ferritin subunits were tested by Western blot analysis. RESULTS: Ferritin concentrations in RA SF were significantly elevated compared to those in osteoarthritis (OA) SF (p < 0.01) and those in RA sera (p < 0.01). Percentages of glycosylated ferritin in SF were low in both RA and OA (RA 11.9 +/- 10.7, n = 41; OA 6.9 +/- 11.0, n = 10). However, percentages of glycosylated ferritin in RA sera (65.9 +/- 15.0, n = 20) were significantly higher than in RA SF (p < 0.01). Western blot analysis revealed both G subunit (23 kDa) and L subunit ( 19 kDa) in RA sera, although SF ferritin was composed mostly of L subunit. CONCLUSION: Significant differences in ferritin molecule composition were observed between sera and SF from patients with RA, which suggests that in RA most SF ferritin is synthesized locally in the affected joint.     

Analysis of human T-cell antigen receptor variable beta gene usage following vaccination with recombinant HBsAg

Exposure of mammalian oocytes to the protein phosphatase (PP)-1 (PP1) and PP2A inhibitor okadaic acid (OA) stimulates oocyte meiosis. However, treated oocytes do not develop beyond metaphase I (MI), and they display morphological aberrations. Experiments were conducted to define inhibitor treatment conditions for macaque oocytes that would result in germinal vesicle breakdown (GVB) stimulation and completion of meiosis without significant cytoplasmic abnormalities. As described above for OA, continual exposure of macaque oocytes to 50 nM calyculin-a (CL-A) significantly enhanced GVB at 24 h compared to that in controls, and the majority of the treated oocytes displayed cytoplasmic abnormalities. However, transient exposure (10 min) of rhesus macaque oocytes to either 50 nM CL-A or 1.0 microM OA enhanced GVB rates compared to that in controls and did not increase the incidence of cytoplasmic abnormalities. Meiotic maturation from germinal vesicle-intact oocytes to MII was enhanced following transient treatment with CL-A or OA compared to that in controls; however, development from MI to MII occurred at a similar frequency. In vitro-matured oocytes transiently exposed to OA and CL-A were capable of fertilization. In addition, ovarian immunohistochemical analysis revealed that both PP1 and PP2A were present in macaque oocytes. PP1 was localized throughout the cytoplasm with a predominance in the nucleus, whereas PP2A was evenly distributed throughout the cytoplasm with a reduction in the nuclear area. These results taken together-differential developmental responses to inhibitor treatment and intracellular enzyme localizations-may be indicative of multiple regulatory roles of PP1 and/or PP2A during meiosis.