Changes in fibroblast growth factor receptors-1c, -2c, -3c, and -4 mRNA in granulosa and theca cells during ovarian follicular growth in dairy cattle.

The various fibroblast growth factors (FGF) regulate their function via binding to 4 main FGF receptor (FGFR) subtypes and their splice variants, FGFR1b, FGF1c, FGFR2b, FGFR2c and FGFR3c and FGFR4, but which of these FGFR are expressed in the granulosa (GC) and theca cells (TC), the 2 main cell layers of ovarian follicles, or change during follicular development is unknown. We hypothesized that FGFR1c, FGFR2c and FGFR3c (but not FGFR4) gene expression in GC (but not TC) would change with follicular development. Hence, the objective of this study was to determine if abundance of FGFR1c, FGFR2c, FGFR3c, and FGFR4 mRNA change according to follicular size, steroidogenic status, and days post-ovulation during growth of first-wave dominant follicles in Holstein cattle exhibiting regular estrous cycles. Estrous cycles of non-lactating dairy cattle were synchronized, and ovaries were collected on either d 3 to 4 (n = 8) or d 5 to 6 (n = 8) post-ovulation for GC and TC RNA extraction from small (1-5 mm), medium (5.1 to 8 mm) or large (8.1-18 mm) follicles for real-time PCR analysis. In GC, FGFR1c and FGFR2c mRNA relative abundance was greater in estrogen (E2)-inactive (ie, concentrations of E2 < progesterone, P4) follicles of all sizes than in GC from large E2-active follicles (ie, E2 > P4), whereas FGFR3c and FGFR4 mRNA abundance did not significantly differ among follicle types or days post-estrus. In TC, medium E2-inactive follicles had greater FGFR1c and FGFR4 mRNA abundance than large E2-active and E2-inactive follicles on d 5 to 6 post-ovulation whereas FGFR2c and FGFR3c mRNA abundance did not significantly differ among follicle types or day post-estrus. In vitro experiments revealed that androstenedione increased abundance of FGFR1c, FGFR2c and FGFR4 mRNA in GC whereas estradiol decreased FGFR2c mRNA abundance. Neither androstenedione nor estradiol affected abundance of the various FGFR mRNAs in cultured TC. Taken together, the findings that FGFR1c and FGFR2c mRNA abundance was less in GC of E2-active follicles and FGFR1c and FGFR4 mRNA was greater in TC of medium inactive follicles at late than at early growing phase of the first dominant follicle support an anti-differentiation role for FGF and their FGFR as well as support the idea that steroid-induced changes in FGF and their receptors may regulate selection of dominant follicles in cattle.

Comparison of effects of four weaning methods on health and performance of beef calves.

Weaning of beef calves is a stressful event that negatively impacts health and performance. A variety of interventions have been proposed to reduce stress and improve gains following weaning. This study used 288 7- to 8-month-old calves from two separate locations, to examine four different weaning strategies, as well as the impact of shipment. Calves were blocked by weight and sex, and then randomly assigned to one of four treatments: abrupt weaning (AW), where calves were separated from the dam on day 0 (D0) and allowed no further contact with the dam; fence line (FL), where calves were weaned on D0 but had fence line contact with dams for 7 days; nose flap (NF), where on day -6 calves received a nose flap that interferes with suckling, then had the flap removed and were weaned from the dam on D0; and intermittent separation (SEP), where calves were removed from dams for 24-h intervals on day -13 and day -6, then weaned on D0, but allowed fence line contact with the dam for 7 days. Each treatment group was further divided into two subgroups, one of which was shipped early (D0 for AW, day 7 for others) or shipped later (day 28). Body weight and sickness were recorded for all groups. Results showed a negative impact on gain for early shipping compared to later shipping, and poorer gain in AW calves than most other treatments. Results of the analyses of morbidity were inconclusive. This study found that delayed shipment following FL weaning improves performance under common management conditions for the US cow-calf industry.

G protein-coupled receptor 34 in ovarian granulosa cells of cattle: changes during follicular development and potential functional implications.

Abundance of G protein-coupled receptor 34 (GPR34) mRNA is greater in granulosa cells (GCs) of cystic vs normal follicles of cattle. The present experiments were designed to determine if GPR34 mRNA in granulosa cell [GC] changes during selection and growth of dominant follicles in cattle as well as to investigate the hormonal regulation of GPR34 mRNA in bovine GC in vitro. In Exp. 1, estrous cycles of nonlactating cows were synchronized and then ovariectomized on either day 3-4 or 5-6 after ovulation. GPR34 mRNA abundance in GC was 2.8- to 3.8-fold greater (P < 0.05) in small (1-5 mm) and large (≥8 mm) estrogen-inactive dominant follicles than in large estrogen-active follicles. Also, GPR34 mRNA tended to be greater (P < 0.10) in F2 than F1 follicles on day 3-4 postovulation. In Exp. 2-7, ovaries were collected at an abattoir and GC were isolated and treated in vitro. Expression of GPR34 was increased (P < 0.05) 2.2-fold by IGF1. Tumor necrosis factor (TNF)-α decreased (P < 0.05) the IGF1-induced GPR34 mRNA abundance in small-follicle GC, whereas IGF1 decreased (P < 0.05) GPR34 expression by 45% in large-follicle GC. Treatment of small-follicle GC with either IL-2, prostaglandin E2 or angiogenin decreased (P < 0.05) GPR34 expression, whereas FSH, cortisol, wingless 3A, or hedgehog proteins did not affect (P > 0.10) GPR34 expression. In Exp. 6 and 7, 2 presumed ligands of GPR34, L-a-lysophosphatidylserine (LPPS) and LPP-ethanolamine, increased (P < 0.05) GC numbers and estradiol production by 2-fold or more in small-follicle GC, and this response was only observed in IGF1-treated GC. In conclusion, GPR34 is a developmentally and hormonally regulated gene in GC, and its presumed ligands enhance IGF1-induced proliferation and steroidogenesis of bovine GC.

Changes in fibroblast growth factor 9 mRNA in granulosa and theca cells during ovarian follicular growth in dairy cattle.

Fibroblast growth factor 9 (FGF9) has been suggested to act as an antidifferentiation factor in cattle by reducing steroidogenesis and increasing cell proliferation in granulosa (GC) and theca (TC) cells. The objective of this study was to characterize FGF9 mRNA abundance in GC and TC during development of dominant follicles in dairy cattle. Estrous cycles of nonlactating dairy cattle were synchronized, and ovaries were collected on either d 3 to 4 (n=8) or 5 to 6 (n=8) postovulation for GC and TC RNA extraction from small (1-5mm), medium (5.1-8mm), and large (8.1-18mm) follicles for PCR analysis. The FGF9 mRNA abundance was greater in GC than in TC. In GC, FGF9 mRNA abundance was greater in small, medium, and large estrogen-inactive [i.e., concentrations of estradiol (E2)P4) follicles at both early (d 3-4) and late (d 5-6) growing phases of first dominant follicle. Abundance of FGF9 mRNA increased in medium-sized follicles from early to late growing phase of the dominant follicle. In TC, FGF9 mRNA abundance was greater in large E2-inactive follicles than in large E2-active follicles on d 3 to 4 postovulation; no significant differences in TC FGF9 mRNA existed among follicle types on d 5 to 6 postovulation. Correlations among levels of follicular fluid hormones and FGF9 mRNA levels revealed significant negative correlations between GC FGF9 mRNA abundance and follicular fluid E2 (r=-0.68), free IGF-1 (r=-0.63), and E2-to-P4 ratio (r=-0.58). In summary, abundance of FGF9 mRNA in GC and TC increases in medium-sized follicles during development of dominant follicles and is less in dominant E2-active than subordinate E2-inactive follicles, suggesting that FGF9 signaling could contribute to normal follicle development and steroidogenesis in dairy cattle.

Changes in brain ribonuclease (BRB) messenger RNA in granulosa cells (GCs) of dominant vs subordinate ovarian follicles of cattle and the regulation of BRB gene expression in bovine GCs.

Brain ribonuclease (BRB) is a member of the ribonuclease A superfamily that is constitutively expressed in a range of tissues and is the functional homolog of human ribonuclease 1. This study was designed to characterize BRB gene expression in granulosa cells (GCs) during development of bovine dominant ovarian follicles and to determine the hormonal regulation of BRB in GCs. Estrous cycles of Holstein cows (n = 18) were synchronized, and cows were ovariectomized on either day 3 to 4 or day 5 to 6 after ovulation during dominant follicle growth and selection. Ovaries were collected, follicular fluid (FFL) was aspirated, and GCs were collected for RNA isolation and quantitative polymerase chain reaction. Follicles were categorized as small (1-5 mm; pooled per ovary), medium (5-8 mm; individually collected), or large (8.1-17 mm; individually collected) based on surface diameter. Estradiol (E2) and progesterone (P4) levels were measured by radioimmunoassay (RIA) in FFL. Abundance of BRB messenger RNA (mRNA) in GCs was 8.6- to 11.8-fold greater (P < 0.05) in small (n = 31), medium (n = 66), and large (n = 33) subordinate E2-inactive (FFL E2 < P4) follicles than in large (n = 16) dominant E2-active (FFL E2 > P4) follicles. In the largest 4 follicles, GCs BRB mRNA abundance was negatively correlated (P < 0.01) with FFL E2 (r = -0.65) and E2:P4 ratio (r = -0.46). In experiment 2, GCs from large (8-22 mm diameter) and small (1-5 mm diameter) follicles were treated with insulin-like growth factor 1 (IGF1; 0 or 30 ng/mL) and/or tumor necrosis factor alpha (0 or 30 ng/mL); IGF1 increased (P < 0.05) BRB mRNA abundance, and tumor necrosis factor alpha decreased (P < 0.001) the IGF1-induced BRB mRNA abundance in large-follicle GCs. In experiment 3 to 6, E2, follicle-stimulating hormone, fibroblast growth factor 9, cortisol, wingless 3A, or sonic hedgehog did not affect (P > 0.10) abundance of BRB mRNA in GCs; thyroxine and luteinizing hormone increased (P < 0.05), whereas prostaglandin E2 (PGE2) decreased (P < 0.05) BRB mRNA abundance in small-follicle GCs. Treatment of small-follicle GCs with recombinant human RNase1 increased (P < 0.05) GCs numbers and E2 production. In conclusion, BRB is a hormonally and developmentally regulated gene in bovine GCs and may regulate E2 production during follicular growth in cattle.

Expression of Ro/SS-A antigen in human skin and heart.

Using human sera monospecific for anti-Ro/SS-A antibodies in an indirect immunofluorescence assay, Ro/SS-A antigen was found to be present in nuclei of human fetal ectodermal cells and newborn epidermal keratinocytes, but not in adult epidermal keratinocytes. After in vitro cultivation, Ro/SS-A antigen was also found in the nuclei of adult skin explant outgrowth cells. The localization of Ro/SS-A in fetal heart could not be precisely determined (possibly nuclear and membrane). Extracts from fetal skin and heart contained Ro/SS-A antigen which is identical to the Ro/SS-A present in WiL2 cell extract. The molecular weight of Ro/SS-A antigenic peptide present in these extracts is around 60K.

The immunologic diagnosis of chronic active "autoimmune" hepatitis: distinction from systemic lupus erythematosus.

We have evaluated the immunologic characteristics often associated with systemic lupus erythematosus in a series of patients with a variety of different liver diseases. Antibody to double-stranded DNA as measured by the Farr assay was detected frequently in patients with various forms of liver disease. No patient with liver disease, including those with a presumed immunologic etiology, was found to have antibody to double-stranded DNA using more specific assays. Other immunologic phenomena such as the presence of immunofluorescent staining at the dermal-epidermal junction in the lupus band test, circulating immune complexes and the presence of antinuclear antibody were present in a number of patients with different forms of liver disease. The absence of antibody to double-stranded DNA in patients with liver disease suggests that there may be a true immunologic distinction between systemic lupus erythematosus and chronic active ("lupoid") hepatitis.

Molecular characteristics of SS-B/La and SS-A/Ro cellular antigens.

Anti-SS-B/La and anti-SS-A/Ro antibodies coexist in certain patients with connective tissue diseases such as systemic lupus erythematosus or Sjögren's syndrome. The respective antigenic structures with which these autoantibodies bind have not been fully characterized. The present study was conducted to better define these two different cellular antigens. WiL2 cell extracts were used to obtain partially purified SS-B/La and SS-A/Ro antigens. Both were found to be present in most fractions obtained after sequential purification with ammonium sulfate salt precipitation, G-200 gel filtration, DE-52 ion exchange chromatography, and preparative slab gel electrophoresis. However, SS-B/La antigenic activity was also found to be present in some fractions that did not contain detectable SS-A/Ro activity. These findings suggested the existence of two different forms of SS-B/La antigen: one containing the SS-B/La antigen only and the other containing both the SS-B/La and SS-A/Ro antigens. The RNA and protein components of these two ribonuclear protein particles were further defined by immunoprecipitation experiments using 32P-labeled WiL2 cell extract. The SS-B/La antigen was found to be associated with several RNAs while the SS-A/Ro antigen was associated with several other distinct RNAs. Both antibodies precipitated a common 43K molecular weight phosphoprotein. The antigenic peptides of these 2 antibodies were analyzed using an immunoblot system. The SS-B/La antigen was present on a 43K peptide which was unstable and could be degraded to several peptides of lower molecular weight (40K, 38K, 30K), while the SS-A/Ro antigen occurred on a peptide having a molecular weight of about 60K.

The binding of antihistone antibodies to Crithidia luciliae kinetoplasts is growth cycle-dependent.

The Crithidia luciliae immunofluorescence (CLIF) assay is widely used to test for native DNA (nDNA) antibodies in the diagnosis and management of systemic lupus erythematosus. However, sera from patients with drug-induced lupus erythematosus or rheumatoid arthritis, which should not contain nDNA antibodies, occasionally react with the CL kinetoplast. We examined 36 sera from patients with systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, and drug-induced lupus erythematosus, who had positive CLIF tests. All 36 sera were also antinuclear antibody-positive with homogeneous and/or peripheral staining patterns on mouse kidney substrates. After hydrochloric acid extraction of the CL smears to remove histone and other nuclear protein antigens, 14 of the 36 sera no longer produced a positive result on the CLIF test. Ten of these 14 sera again gave a positive CLIF result after the hydrochloric acid-extracted Crithidia substrate had been reconstituted with purified histone. These studies demonstrated that kinetoplast binding was due to antihistone antibodies in at least 10 of 36 initially CLIF-positive sera. Antihistone antibodies were then purified with a histone-affinity column, and these purified antibodies were reactive with CL kinetoplasts. Thus, the CLIF test is not specific for nDNA antibodies. Additional studies using CL from different days of culture indicated that histone antigen expression in the CL kinetoplast was a function of the life cycle of this organism and is most readily detected 2 days after initiation of culture.

Prognostic significance of subepidermal immune deposits in uninvolved skin of patients with systemic lupus erythematosus: a 10-year longitudinal study.

The detection by direct immunofluorescence of subepidermal immune deposits in clinically normal skin of patients with systemic lupus erythematosus has become known as a positive lupus band test (LBT). To gain a better understanding of the relation between the LBT and prognosis in systemic lupus erythematosus (SLE) a prospective longitudinal study has been carried out in 51 SLE patients covering a 10-year period. A total of 223 LBTs were obtained from clinically normal skin of the medial volar forearm on these 51 patients (average, 4.4 per patient) and the results correlated with clinico-pathologic features of the disease and outcome. Findings from the initial LBT (obtained while on no systemic therapy) were used to divide patients into LBT-positive and LBT-negative groups. With the exception of patients subsequently treated with daily doses of prednisone greater than 40 mg or cytotoxic agents, the patients in the LBT-positive group usually remained LBT-positive. The LBT-negative patients usually remained LBT-negative on repeated testing. A comparison of clinical features in the two groups revealed a 55% prevalence of lupus nephropathy in the LBT-positive group as opposed to 23% in the LBT-negative group (p = 0.025). Although the two groups had similar serum creatinine levels at the time of the initial LBT, the maximum serum creatinine (mean, 3.0 mg/dl) in the LBT-positive group was significantly higher than the maximum (mean, 1.2 mg/dl) in the LBT-negative group (p = 0.04). Furthermore, only 9% of renal biopsies in the LBT-negative group showed diffuse proliferative glomerulonephritis in contrast to 65% of biopsies in the LBT-positive group (p = 0.007). Lastly, the two groups were compared with regard to outcome; 10-year survival from the time of diagnosis was 95% in the LBT-negative group as opposed to only 54% in the LBT-positive group (p = 0.007). These findings indicate that a positive LBT has predictive value in that it identifies a subset of SLE patients with more aggressive renal disease and significantly decreased long-term survival.

Reappraisal of the specificity of the Crithidia luciliae assay for nDNA antibodies: evidence for histone antibody kinetoplast binding.

Five different high-titer histone antibody-containing sera were assayed by the Crithidia luciliae indirect immunofluorescence (CLIF) technic. Three of these sera produced kinetoplast binding at titers of 1/40 to 1/80. The kinetoplast binding activity was abolished by HCl acid pretreatment of the Crithidia substrate, suggesting that the kinetoplast binding activity was not due to antibodies against native DNA (nDNA). Histone antibodies were purified from two of the three positive sera by affinity chromatography utilizing purified preparations of histone. Both purified antibody preparations also had kinetoplast-binding activity, confirming that the Crithidia kinetoplast contains histone-like proteins. Therefore, Crithidia luciliae (CL) kinetoplast binding activity does not necessarily indicate the presence of anti-nDNA antibodies. Routinely pretreating the CL substrate with 0.1 N HCl would eliminate the possibility of histone antibody kinetoplast binding in the CLIF assay. Whether such pretreatment would alter the binding of anti-NDNA to the kinetoplast remains to be determined.

Reevaluation of specificity of Crithidia luciliae kinetoplast as a substrate for detecting antibodies to double-stranded deoxyribonucleic acid.

Until now, the kinetoplast of the hemoflagellate Crithidia luciliae has been believed to be a specific indirect immunofluorescence substrate for detecting antibodies to double-stranded deoxyribonucleic acid (DNA). We have recently identified a patient in whom there was the apparent presence of procainamide-induced antinuclear antibodies at a titer of 1/1,280 on mouse kidney sections. This patient's serum also contained antihistone antibodies at a titer of 1:1,280, on the basis of a mouse kidney section histone reconstitution assay. In addition, results of an enzyme-linked immunosorbent assay (ELISA) using a pure histone preparation were strongly positive. We found that this serum produced C. luciliae fluorescence to a titer of 1:80. After pretreatment of the C. luciliae substrate with hydrochloric acid to remove histones, this serum no longer produced kinetoplast fluorescence. However, this serum again produced kinetoplast fluorescence when assayed on a hydrochloric acid-treated C. luciliae substrate that had been reconstituted with purified histone. The negative kinetoplast reactivity after hydrochloric acid treatment of the C. luciliae substrate suggests that the antibody is reacting with acid-extractable nucleic acids, not necessarily the double-stranded DNA, or the antibody is directed against nuclear proteins such as DNA-histone complexes of deoxyribonucleoprotein. The finding that reactivity reappeared after the histone was reconstituted back to the hydrochloric acid-treated C. luciliae substrate suggests that the primary antibody in this serum is directed against DNA-histone complexes or histone alone. These findings strongly suggest that the kinetoplast of C. luciliae contains antigens other than double-stranded DNA and that is not a specific substrate for the detection of double-stranded DNA antibodies by indirect immunofluorescence.

Relationships between antinuclear and anti-Ro/SS-A antibodies in subacute cutaneous lupus erythematosus.

A certain degree of confusion has arisen regarding the relationship between patients having subacute cutaneous lupus erythematosus (SCLE) and those with "antinuclear antibody-negative" systemic lupus erythematosus (ANA-negative SLE). One of the confusing issues relates to published differences in the autoimmune serologic findings of these two patient groups. In order to clarify this issue, we have screened the sera from thirty-seven patients with SCLE for the presence of fluorescent antinuclear antibodies (FANA) on two different substrates--human Hep-2 tissue culture cells and mouse kidney sections. In addition, these same sera were assayed for anti-Ro/SS-A precipitin antibodies. Seventy-eight percent of the sera were FANA-positive at a titer of 1:10 or greater when tested on human Hep-2 cells, whereas 76% were positive at a titer of 1:80 or greater. Fifty-one percent were positive on mouse kidney sections at a titer of 1:10 or greater, whereas 46% were positive at a titer of 1:20 or greater. Twenty-two percent of these sera were completely FANA-negative on both human and mouse substrates. None of these sera that were negative on both substrates contained anti-Ro/SS-A antibodies. However, 69% of the sera that were FANA-positive on both human and mouse substrates were found to have detectable anti-Ro/SS-A antibodies. Ninety percent of the SCLE sera that were FANA-positive on human Hep-2 cells, but negative on mouse kidney sections, contained anti-Ro/SS-A antibodies. These sera gave a speckle-like, or particulate, nuclear immunofluorescence staining pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

Herpes gestationis in blacks.

To our knowledge, only two cases of herpes gestationis (HG) have been previously reported in black patients. We describe herein two black women with confirmed HG. Both had typical historical and clinical features of the disease. Direct immunofluorescence microscopy demonstrated complement deposition along the dermoepidermal junction in both women. Where HLA typing was possible (in one patient), the combination of HLA-DR3 and HLA-DR4 was found. In addition, anti-HLA-DR2 antibodies were present in serum samples taken from this patient. The occurrence of the HLA-DR3-DR4 combination has been reported to be greatly increased in whites with HG (43%, as compared with a 3% incidence in control subjects). The HLA-DR4 antigen is uncommon in American blacks, which may explain the infrequent occurrence of HG in this population. The occurrence of the DR2 antigen is increased in the husbands of women with HG, and this increase is most pronounced in the husbands of patients with the DR3-DR4 combination. The occurrence of positive immunofluorescence microscopy findings, together with the presence of a rare histocompatibility antigen combination previously associated with HG and the presence of anti-DR2 antibodies in the serum of one of our patients all suggest that HG is pathogenically identical in both blacks and whites.

Subacute cutaneous lupus erythematosus and discoid lupus erythematosus. Comparative histopathologic findings.

Subacute cutaneous lupus erythematosus (SCLE) is a recently described distinct subset of lupus erythematosus (LE) having characteristic clinical, serologic, and genetic findings. This study describes the histopathologic characteristics of SCLE and determines whether it could be differentiated from discoid lupus erythematosus (DLE) on histopathologic grounds alone. Biopsy specimens from 33 patients having either SCLE or DLE, as defined by strict clinical criteria, were examined without knowledge of the clinical diagnosis. Histologic discrimination between SCLE and DLE was accomplished in 82%. The specimens from DLE lesions had substantially more hyperkeratosis, basement membrane thickening, follicular plugging, and superficial and deep inflammatory cell infiltrate, while SCLE had more epidermal atrophy. The histopathologic differences between SCLE and DLE further support the concept that SCLE is distinct from DLE and should be considered a unique subset of LE.

Perturbation of epidermal Langerhans cells in immunosuppressed human renal allograft recipients.

As an initial attempt to gain a better understanding of the basis for the increased incidence of ultraviolet-light-related skin cancer in chronically immunosuppressed human renal allograft recipients, we have compared both morphological and functional characteristics of epidermal Langerhans cell (LC) populations present in the forearm skin of nine such patients with those of age, sex, and race-matched controls. The LC surface densities in vacuum-induced blister-derived epidermal sheets taken simultaneously from extensor and flexor forearm skin of the patients were significantly lower than those observed in the controls. The most abnormal LC densities seen were in the patients' extensor forearm skin. Likewise there were disturbances in LC distribution and morphology that were most marked in the extensor forearm skin of patients. Differences in the alloantigen-presenting capacity of LCs present in epidermal cell suspensions prepared from patient and control forearm skin were also noted--however, these differences were not as great as were the LC density differences. The alloantigen-presenting capacity of patients' LCs was depressed proportionately more than was the alloantigen presenting capacity of their peripheral blood mononuclear cells. These results demonstrate that the LC population is clearly perturbed in human renal allograft recipients and that this perturbation is greatest in a sun-exposed region of skin.

Cutaneous mast cell maturation does not depend on an intact bone marrow microenvironment.

We sought to determine whether the maturation of murine cutaneous mast cells from stem cells depends on an intact bone marrow microenvironment. Normal bone marrow cells (+/+) were infused into 2 groups of mast cell-deficient mice: WBB6F1-W/Wv mice and 89Sr-pretreated W/Wv mice. 89Sr is a long-lived bone-seeking radioisotope which provides continuous irradiation of the marrow and thereby ablates the marrow microenvironment. Skin biopsies revealed that the 89Sr-pretreated mice and the controls had repopulated their skin with mast cells equally well. Natural killer cell function was significantly depressed in the 89Sr-treated mice, confirming that the marrow microenvironment had been functionally altered. We conclude that, although the precursors for cutaneous mast cells are marrow derived, they do not need an intact marrow microenvironment for maturation.

Comparison of substrates for the detection of antinuclear antibodies in normals and in patients with connective tissue and other diseases.

There has been some controversy regarding the relative merits of cell lines versus frozen tissue substrates for the detection of antinuclear antibodies (ANA) by indirect immunofluorescence. We have compared two cell lines (KB and HEP2) with frozen mouse kidney for the detection of ANA in several groups of individuals. Cell lines were more likely to detect ANA than frozen mouse kidney in normal individuals and in hospital and clinic patients with diseases other than connective tissue diseases when sera were examined at manufacturer's recommended screening dilutions. There was also a trend for the cell lines to demonstrate ANA more frequently than mouse kidney in patients with systemic lupus erythematosus and other connective tissue diseases, but the differences were not statistically significant. Centromere antibodies could be reliably suspected only on cell lines and could be confirmed only if mitotic figures were present.

Prevention of lethal, minor-determinate graft-host disease in mice by the in vivo administration of anti-asialo GM1.

Graft-vs-Host disease (GVHD) remains a devastating problem in human bone marrow transplantation (1, 2). Because removal of Thy-bearing cells from the donor inoculum has prevented GVHD in murine models (3, 4), it has been hoped that a similar cell surface antigen or combination of antigens could be found in humans. Unfortunately, treatment of human donor cells with various T cell antisera has not yet been successful in preventing GVHD (5). Encouraging results have been reported in five patients who received bone marrow depleted of T cells by the sequential use of soybean agglutinin and the differential sedimentation of cells forming rosettes with sheep red blood cells (6). Although donor T cells are thought to be necessary for initiating GVHD, the immunopathogenesis of GVHD is still not understood. Because donors and recipients are routinely major histocompatibility complex matched and chosen to be nonreactive in mixed lymphocyte cultures human GVHD is thought to result from minor histocompatibility antigen disparities. Lopez and coworkers (7, 8) found a strong association between the incidence of human GVHD and the pretransplant levels of natural killer (NK) activity of the recipients; when the recipient NK activity was low, GVHD rarely developed. They speculated that the NK cell lineage is serving as an important stimulator-inducer. We therefore examined the in vivo effects of anti-asialo GM1 on a murine model of GVHD based on minor antigen disparity. This antiserum has several immunologic effects, including a profound NK suppression. We found that the mice treated with this antibody have normal survival rates, even though they do develop histologic GVHD in the skin. This finding suggests the possibility of a new prophylactic approach to human GVHD and raises many questions regarding the function of asialo GM1-bearing cells in immune regulation.

Paternal histocompatibility (HLA) antigens and maternal anti-HLA antibodies in herpes gestationis.

It has been suggested that anti-HLA antibodies might be involved in the pathogenesis of herpes gestationis (HG). Accordingly, we have studied the frequency and specificity of such antibodies in 26 female patients with immunologically proven HG. In addition, to further investigate the potential association of the husband's antigens in the development of this disorder, we have performed HLA typing in 20 of the husbands of these women. HLA-DR2 was found in 50% of the husbands (controls 25%, p = 0.04). The increase was more pronounced in the husbands of patients with the HLA-DR3, DR4 combination (64%, p less than .01) than in the husbands of those with other antigen combinations. Anti-HLA antibodies were found in 85% of women with a history of HG. Approximately 25% of normal, multiparous women have such antibodies in their sera. In normal women, their presence has not been associated with increased fetal or maternal risk. These data suggest that the husband's HLA type may be associated with the development of HG in the wife. Anti-HLA antibodies are present in most patients, but their significance remains uncertain.