Autoantibodies encoded by the most Jh-proximal human immunoglobulin heavy chain variable region gene.

Little is known about the utilization of human Ig heavy chain variable gene segments (VH segments) in different B-lineage cell populations or in antibodies of particular specificity and function. We now demonstrate that human antibodies with Ig VH regions encoded by the most JH-proximal human VH segment (VH6) have specificities resembling those of autoantibodies present in sera of patients with systemic lupus erythematosus (e.g., anti-DNA and anticardiolipin). These specificities appear to be encoded by the germline VH6 gene because the activity was found in multiple independent VH6 antibodies in which the light chain varied with respect to isotype and V kappa subgroup. Features of CDR3 length and somatic mutation patterns in several VH6 antibodies suggested that they were selected by the immune system.

Quantification of viable granulosa cells in murine ovarian follicles.

Ovarian follicle growth and oocyte maturation depend on the viability of granulosa cells (GC). We quantified GC in whole mouse follicles. Single follicles were isolated from adult mouse ovaries and stained with DAPI or Live-Dead stain before fixation. An objective image analysis protocol for counting fluorescent labeled GC was developed that used Image J software to measure GC cytoplasmic and nuclear areas. These data were compared to the number of GC obtained by disaggregating 96 follicles with enzymes to produce a suspension of GC, which then was stained with trypan blue and assessed using a hemocytometer. We found a linear relation between GC/follicle and follicle diameter. Viability of GC/follicle ranged from 40 ± 11 to 72 ± 7%. The coefficient of variation for image analysis of DAPI stained GC by different assessors was 4%, but the number of GC obtained from image analysis was approximately 50% less than from disaggregated follicles. The number of GC in intact mouse follicles was greater than the number reported earlier for fixed ovarian sections. We found that the number of GC was less in fluorescence labeled follicles; it is possible that the three-dimensional structure of the intact follicles obscured the fluorescent signal. Direct quantification of viable GC isolated from follicles appears to be the most accurate method.

The antioxidant beta-carotene prevents covalent cross-linking between cholesterol side-chain cleavage cytochrome P450 and its electron donor, adrenodoxin, in bovine luteal cells.

Steroid hormones are an important class of hormones synthesized from cholesterol by a number of endocrine organs; including ovaries, placenta, testes and adrenal glands. The first and rate-limiting step in steroidogenesis is the cleavage of the side-chain of the cholesterol molecule, catalysed by a cytochrome P450 enzyme, cholesterol side-chain cleavage enzyme. This enzyme, as with other P450 enzymes, produces oxygen radicals. Oxygen free radicals can cause deleterious effects such as cross-linking and aggregation of proteins. Cells can protect against such damage with the use of antioxidants. The corpus luteum, or 'yellow body', of the ovary is very steroidogenic and is exceedingly rich in the yellow antioxidant, beta-carotene. The corpus luteum produces the steroid hormone progesterone that is needed to support pregnancy. Here we have shown that by depleting, or conversely repleting, luteal cells of their beta-carotene content in vitro that P450 side-chain cleavage enzyme became covalently non-disulfide cross-linked to its electron donor, adrenodoxin, and hence inactivated. Bovine luteal cells were cultured in 10% fetal calf serum with or without additional treatments for up to 72 h. Under control conditions the cellular levels of beta-carotene and alpha-tocopherol fell by 50% within 24 h and remained low. P450 side-chain cleavage enzyme become non-disulfide covalently cross-linked to its electron donor, adrenodoxin, as determined by Western immunoblotting (N = 18). Aminoglutethamide inhibited this cross-linking. The addition of beta-carotene at levels found in bovine serum, but not alpha-tocopherol or ascorbic acid, inhibited the degree of the cross-linking.(ABSTRACT TRUNCATED AT 250 WORDS)

The physiology of the ovary: maturation of ovarian granulosa cells and a novel role for antioxidants in the corpus luteum.

During folliculogenesis the granulosa cells divide whilst in contact with each other, and so exhibit some of the characteristics of stem cells. In vitro we have shown that bovine granulosa cells from 3-7 mm follicles, like stem cells, divide without the need for a substratum, and produce colonies of cells. Growth factors, bFGF and IGF's, stimulate their division. These cells secrete and assemble a basal lamina, suggesting that the follicular basal lamina is produced by the granulosa cells. They have the morphological characteristics of follicular granulosa cells. Thus this system is ideal for studying the functions of immature granulosa cells because the cells do not spontaneously differentiate or luteinize into luteal cells, as occurs in culture on a substratum. On differentiation into luteal cells in vivo the cells express the steroidogenic enzymes for progesterone production and accumulate beta-carotene. During culture of bovine luteal cells we observed that a proportion of the steroidogenic enzyme cholesterol side-chain cleavage cytochrome P450 enzyme became chemically cross-linked to its electron donor, adrenodoxin. P450 enzymes produce oxygen free radicals and oxygen free radicals can cause cross-linking between proteins in close proximity. Cell protect against this damage by the use of antioxidant vitamins. Repleting the cultured luteal cells with beta-carotene reduced the amount of cross-linking. We conclude that the high levels of beta-carotene in corpora lutea are to protect against damage due to oxygen free radicals generated in the course of progesterone synthesis.

Concentrations of cytochrome P450 cholesterol side-chain cleavage enzyme and 3 beta-hydroxysteroid dehydrogenase during prostaglandin F2 alpha-induced luteal regression in cattle.

Prostaglandin F2 alpha (PGF2 alpha)-induced regression of the corpus luteum causes both plasma progesterone concentrations and luteal concentrations of mRNA encoding the steroidogenic enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) to fall in parallel. To investigate the hypothesis that a decline in the concentrations of mRNA encoding steroidogenic enzymes causes plasma progesterone to fall, the luteal concentrations of the enzymes 3 beta-HSD and cytochrome P450 cholesterol side-chain cleavage were measured during induced luteolysis. Holstein heifers were treated with PGF2 alpha (25 mg Lutalyse) on Day 6 or Day 7 of the oestrous cycle and corpora lutea were collected 0 h, 2 h, 12 h, and 24 h later (n = 6, 4, 4, and 4 respectively). Analyses of the steroidogenic enzymes were carried out by Western immunoblotting. The luteal concentrations of both steroidogenic enzymes did not decrease over the 24-h period. It is concluded that, although the concentrations of mRNA encoding steroidogenic enzymes may decline in response to PGF2 alpha, this does not lead to a sufficiently rapid reduction in the concentrations of the enzymes to precede, and thus cause, the decline in plasma progesterone concentrations. Thus, the mechanism for the initial decline in plasma progesterone concentrations during luteolysis is still not known.

Emergency surgical admissions in patients aged more than 80 years: a study over four decades.

BACKGROUND: The proportion of older patients in the community is rising. The aim of this study was to determine the trend in emergency surgical admissions in patients over 80 years of age in 1997 compared with the previous three decades. PATIENTS AND METHODS: Data were obtained on all patients over 80 years of age admitted as general surgical emergencies in 1997 to the Royal Berkshire and Battle Hospitals, Reading, UK. Reasons for admission, management, mortality and duration of hospital stay were recorded and compared with results from 1966, 1976 and 1989. RESULTS: During 1997, 4807 patients over the age of 80 years were admitted as emergencies to all specialities. Of these, 447 (9.3%) were surgical. This compares with 122 in 1966, 248 in 1976 and 339 in 1989. Emergency surgical workload in patients over 80 years of age had increased from 6.2% in 1966 to 12% in 1997. A random sample of 261 patients was analysed. In-patient mortality was 13.8% in 1997 compared with 21.8% for 1976 and 22.4% for 1989. Median length of stay was 8 days (range, 0-41 days) for 1997 and 1989 compared with 14 days in 1976. Twenty-four patients either needed admission to other specialities or need not have been admitted as emergencies at all and were classified as inappropriate admissions to the general surgical ward. CONCLUSIONS: The trend of increased number of patients over the age of 80 years being admitted as emergencies to general surgery continues through four decades. There has been a decrease in mortality and length of stay since 1966, but no decrease in length of stay in 1997 compared with 1989. Avoiding inappropriate admissions would result in a significant improvement in bed utilisation for elective surgery and help to reduce waiting lists.

Ubiquitin and apoptosis in the corpus luteum of the marmoset monkey (Callithrix jacchus).

The polypeptide ubiquitin covalently binds to cytoplasmic proteins and marks them for proteolytic degradation. Ubiquitin is upregulated during apoptosis in some systems. Apoptosis increases during luteolysis but it is not known whether ubiquitin is expressed in regressing corpora lutea. Marmoset ovaries were removed on day 10 of the luteal phase from animals that had received either no treatment, treatment with the PGF2 alpha analogue cloprostenol 24 h earlier, or treatment with the GnRH antagonist antarelix for either 24 or 48 h before ovary collection. Ubiquitin was localized on ovarian sections by immunocytochemistry, and oligonucleosome formation characteristic of apoptosis was examined in isolated corpora lutea by electrophoresis of extracted [32P]DNA. Oligonucleosome formation was low in midluteal corpora lutea on day 10 but increased after induced luteal regression with PGF2 alpha and GnRH antagonist. Nuclear ubiquitin immunoreactivity was found in 1.66 +/- 0.66 steroidogenic cells and cytoplasmic staining was found in 0.4 +/- 0.3 steroidogenic cells (per x 40 field of view) in midluteal phase corpora lutea on day 10. Luteolytic induction with PGF2 alpha significantly increased the number of cells exhibiting cytoplasmic immunoreactivity to 12.24 +/- 1.6 (P < 0.05). Ubiquitin immunoreactivity was not observed after GnRH-induced luteal regression. Apoptotic oligonucleosome formation was found after induced luteal regression with both PGF2 alpha and GnRH antagonist, but ubiquitin upregulation only occurred after PGF2 alpha-induced regression. These results indicate that ubiquitin expression is not specific for luteolysis and is not an indicator of luteal apoptosis, but that the polypeptide does play a role in luteal cellular responses to PGF2 alpha.

Induced luteolysis in the primate: rapid loss of luteinizing hormone receptors.

The molecular mechanisms involved in luteolysis are still unclear in the primate. This study aimed to investigate the effect of induced luteolysis on the ovarian luteinizing hormone (LH) receptor and the steroidogenic enzyme, 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in the marmoset monkey. Luteolysis was induced in the mid-luteal phase either directly by systemic prostaglandin F2alpha (PGF2alpha), or indirectly by LH withdrawal using systemic gonadotrophin releasing hormone antagonist (GnRHant) treatment. The LH receptor was studied by isotopic mRNA in-situ hybridization and in-situ ligand binding and 3beta-HSD expression was studied using isotopic mRNA in-situ hybridization and immunohistochemistry. Induced luteolysis was associated with a reduction in the expression of LH receptor (P < 0.0001) and 3beta-HSD mRNA, closely followed by a reduction in the LH receptor (P < 0.05) and 3beta-HSD protein concentrations within 24 h. There were no differences in the findings whether luteolysis was induced with PGF2alpha or GnRHant. This study shows that disparate mechanisms to induce luteolysis in the primate result in an identical rapid loss of the LH receptor and 3beta-HSD. In conclusion, induced luteolysis leads to rapid loss of the steroidogenic pathway in luteal cells.

Cell proliferation and vascular morphology in the marmoset corpus luteum.

Luteal formation is associated with angiogenesis and low progesterone production. Maximal mid-luteal phase progesterone production is concurrent with extensive vascularization, and luteolysis occurs when steroidogenesis decreases. Angiogenic cell proliferation and vascular changes have not been examined in the marmoset. The aim of this study was to examine vascular morphology throughout the luteal phase by identifying: (i) von Willebrand factor VIII antigen (vW)-immunopositive endothelial cells; (ii) Ki67-positive proliferating cells; and (iii) bromodeoxyuridine-positive proliferating cells. Marmoset corpora lutea were examined throughout the cycle, and natural regression was compared with induced luteolysis after administration of a prostaglandin F(2alpha) analogue or gonadotrophin-releasing hormone (GnRH) antagonist. Steroidogenic and endothelial cells were positive for proliferation markers. Endothelial cell proliferation was highest during luteal formation, then decreased and remained low during the luteal phase and functional regression, however endothelial cell proliferation increased during structural regression. Endothelial cell proliferation was unchanged by induced regression. The area of vW immunostaining was highest during luteal formation, decreased thereafter and remained constant during the luteal phase and regression. Distribution of immunostaining indicated the presence of an extensive capillary network, but during structural regression the numbers of capillaries decreased and numbers of microvessels increased. These results suggest that vascular changes are concurrent with changes in the functional status of the marmoset corpus luteum.

Endothelial cell proliferation follows the mid-cycle luteinizing hormone surge, but not human chorionic gonadotrophin rescue, in the human corpus luteum.

The corpus luteum is essential for the maintenance of early pregnancy in women. Angiogenesis may be one factor involved in luteal rescue. The aim of this study was to determine the changes in endothelial cell proliferation throughout the luteal phase and in human chorionic gonadotrophin (HCG)-simulated early pregnancy. Human corpora lutea obtained throughout the luteal phase and in simulated early pregnancy were immunostained with antibodies for endothelial and proliferating cells. Number and distribution of endothelial and proliferating cells were examined. Endothelial cells were least abundant in the early luteal phase, increasing in the mid-luteal phase (P < 0.03). Endothelial numbers did not differ significantly between the late and the rescued corpora lutea. Endothelial cell proliferation was greatest in the early luteal phase and continued at a lower level during later stages. Simulated early pregnancy resulted in no change in endothelial cell proliferation. These results showed that a high degree of endothelial cell proliferation is associated with formation of the human corpus luteum. Unchanging levels of proliferation following HCG treatment (for 5-8 days from day 12 to day 16 post-ovulation, at 125 IU to 16,000 IU, following a daily doubling of dose) suggest that alternative processes are involved during luteal rescue.

Cell death during luteal regression in the marmoset monkey (Callithrix jacchus).

The mechanism controlling luteal regression in primates is unknown but may involve cell death by apoptosis. Marmoset ovaries containing corpora lutea were studied at different stages of the normal ovarian cycle. Two additional groups of animals underwent induced luteolysis with either the prostaglandin F2 alpha analogue, cloprostenol, or the GnRH antagonist, antarelix, at the mid-luteal phase. Apoptosis in ovarian sections was estimated both by counting the number of cells exhibiting morphological features of apoptosis and by in situ labelling the 3' ends of the DNA fragments with digoxigenin-11-dUTP. Apoptosis was found to be significantly increased in corpora lutea in the early follicular phase (equivalent to the later stage of luteal lifespan) compared with the mid-luteal phase corpora lutea, as judged by either computerized morphometry or 3' end labelling. Apoptosis was also increased by the administration of either cloprostenol or antarelix when using the 3' end labelling end point, but only after cloprostenol when using computerized morphometry. A further form of cell death, characterized by the formation of cytoplasmic vacuoles, was also observed in corpora lutea undergoing both induced and spontaneous regression. These results demonstrate that apoptosis within the primate corpus luteum is increased in both physiological and induced luteal regression. In addition, they show that an alternative form of cell death is involved in both spontaneous and induced luteal regression, although the relative importance of the two mechanisms remains to be determined.

Frequency of VH-gene utilization in human EBV-transformed B-cell lines: the most JH-proximal VH segment encodes autoantibodies.

We have studied VH-gene utilization in a collection of 187 IgM-secreting EBV-transformed cell lines and have begun to correlate VH-gene family expression with binding properties of the secreted immunoglobulins. The results of these studies demonstrate that (1) frequency of VH-gene utilization in fetal and adult tissue-derived cell lines correlates with the complexity of the family and (2) the single-membered most JH-proximal VH-6 family encodes autoantibodies reminiscent of autoantibodies found in the sera of patients with systemic lupus erythematosus. Nucleotide sequence analysis of VH-6-expressing clones revealed that each clone utilizes a short DH segment, resulting in a CDR3 region of conserved length. Our data suggest that EBV does not selectively transform human B cells on the basis of VH-gene family expression and that the VH-6 family encodes polyspecific autoantibodies that may serve an important regulatory function in the immune system.

Tetracyclines inhibit activated B cell function.

Tetracyclines have recently been shown to exert a number of pleiotropic anti-inflammatory and immunomodulatory activities, independent of their antibiotic properties. These include the ability to inhibit metalloproteinases (MP), a class of enzymes involved in crucial cellular functions such as the shedding of soluble mediators and their receptors from the cell surface, as well as interaction with, and remodeling of, the extracellular matrix. Here we report that doxycycline at therapeutic concentrations (1--5 microg/ml) significantly suppresses Ig secretion and class switching by in vitro activated murine B cells. Suppression of Ig secretion correlates with a decrease in levels of mRNA for the terminal B cell differentiation-associated genes Blimp-1 and mad-4, as well as to a reduction in expression of the plasma cell markers Syndecan-1 and J chain. Inhibition of class switching occurs at the recombination stage and is also induced by other MP inhibitors, including tetracycline analogs lacking antibiotic activity and the chemically unrelated hydroxamate KB8301. These novel, direct effects of MP inhibitors on B lymphocytes suggest an intrinsic role for MP in B cell activation and likely explain some of the observed in vivo immunomodulatory properties of tetracyclines. Moreover, these findings have significant implications for tetracycline therapy in Ig-mediated autoimmune or allergic diseases and raise questions about the use of doxycycline-inducible transgenic systems for the study of B cell function.