Temperature-mediated germ cell loss in the testis is associated with altered expression of the cell-cycle regulator p53.

The dependence of spermatogenesis on the relatively cool environment of the scrotum is well known, and recent work has shown that germ cells undergo apoptosis upon exposure to abdominal temperature. p53 is a potent inducer of apoptosis and regulator of cell growth, and is found in high concentrations in the testis. The purpose of this study was to determine whether exposure of the testes to suprascrotal temperature was associated with alterations in testicular p53 expression. Male adult CD-1 mice were rendered unilaterally cryptorchid by surgically fixing one testis to the anterior abdominal wall while leaving the contralateral tests in the scrotum to serve as the euthermic control. p53 expression was evaluated in the cytoplasmic, soluble nuclear, and insoluble fractions by Western blot analysis with the monoclonal p53 antibody pAb240. The weights of the scrotal testes were unchanged over the 15 day study period. The weights of the cryptorchid testes remained stable for 7 days and then decreased by approximately 40% over the next two days. Histological evidence of germ cell loss was evident only after day 7. Altered expression of p53 protein in the cryptorchid testis was noted beginning on day 7, and consisted of the expression of a new 47 kD isoform of p53 in the cytosolic form and a 30 kD isoform in the soluble nuclear fraction. Scrotal testes showed no changes at any time point. These results demonstrate altered expression of the regulatory protein p53 beginning 1-2 days prior to the onset of germ cell loss following experimental unilateral cryptorchidism. Given the known function of p53 as an inducer of apoptotic cell death, these observations suggest a significant role for p53 in temperature-mediated germ cell loss.

Ureteral pseudodiverticulosis: the case for the retrograde urogram.

Ureteral pseudodiverticula are small outpouchings along the length of the ureter diagnosed primarily by the retrograde urogram. They are associated with hematuria and urinary tract infections, although it is now known whether they cause these associated symptoms or are a result of them. Herein we report 2 cases that highlight the characteristic clinical correlates of ureteral pseudodiverticulosis and review the pertinent literature.

Anti-equine tumor necrosis factor (TNF) activity of antisera raised against human TNF-alpha and peptide segments of human TNF-alpha.

Antisera raised in rabbits against either purified recombinant-derived human tumor necrosis factor (TNF)- alpha (huTNF) or huTNF peptide-bovine thyroglobulin conjugates were evaluated for anti-equine TNF (eqTNF) activity. Binding and neutralizing anti-eqTNF activities were found in antisera raised against whole huTNF or against either of the peptides containing the N-terminal 15 amino acids of huTNF (huTNF[1-15] and huTNF[1-31]). Anti-eqTNF activity was not detected in antisera raised against huTNF[65-79], huTNF[98-111] or huTNF[124-141] peptides. The addition of excess huTNF[1-15] completely inhibited the ability of anti-huTNF[1-15] to bind eqTNF and reduced by approximately 25% the anti-eqTNF activity of an antiserum raised against whole huTNF. Nonconjugated huTNF[1-15] did not have eqTNF agonist or antagonist activity. Results were consistent with previous structural and functional data implicating the N-terminus of huTNF in receptor binding and indicate that the homologue of huTNF[1-15] on eqTNF may be a potentially important target for neutralizing anti-eqTNF antibodies.

Tumor necrosis factor and endotoxin can cause neutrophil activation through separate pathways.

We investigated the possibility that tumor necrosis factor (TNF) mediates neutrophil activation by endotoxin. The number of C3b receptors on the neutrophil cell-surface was used as the indicator of activation, as assessed by indirect immunofluorescence. Incubation of buffy-coat neutrophils with TNF-alpha for 30 minutes at 37 degrees C caused neutrophil activation, increasing C3b receptor-dependent fluorescence from 340 with buffer alone to 580 with TNF (250 pg/mL). Increasing amounts of anti-TNF IgG progressively inhibited neutrophil activation by TNF (250 pg/mL). Addition of the active dose range of anti-TNF to neutrophils incubating in endotoxin (10 ng/mL) did not affect the degree of endotoxin-mediated neutrophil activation. Mixtures of neutrophils with the 50% suppressive dose of anti-TNF and varying endotoxin concentrations showed the same degree of neutrophil activation as mixtures without the antibody. Thus, an antibody that can inhibit TNF-mediated neutrophil activation does not inhibit endotoxin-mediated neutrophil activation. We conclude that endotoxin and TNF can activate neutrophils through separate pathways.

Antisera and monoclonal antibodies specific for epitopes generated during oxidative modification of low density lipoprotein.

Increasing evidence indicates that low density lipoprotein (LDL) has to be modified to induce foam cell formation. One such modification, oxidation of LDL, generates a number of highly reactive short chain-length aldehydic fragments of oxidized fatty acids capable of conjugating with lysine residues of apoprotein B. By immunizing animals with homologous malondialdehyde-modified LDL (MDA-LDL), 4-hydroxynonenal-LDL (4-HNE-LDL), and Cu+(+)-oxidized LDL, we developed polyvalent and monoclonal antibodies against three epitopes found in oxidatively modified LDL. The present article characterizes an antiserum and monoclonal antibody (MAL-2 and MDA2, respectively) specific for MDA-lysine, and an antiserum and monoclonal antibody (HNE-6 and NA59, respectively) specific for 4-HNE-lysine. In addition, a monoclonal antibody (OLF4-3C10) was developed against an as yet undefined epitope generated during Cu++ oxidation of LDL. With these antibodies, we demonstrated that MDA-lysine and 4-HNE-lysine adducts develop on apo-lipoprotein B during copper-induced oxidation of LDL in vitro. The application of these antibodies for immunocytochemical demonstration of oxidized lipoproteins in atherosclerotic lesions of progressive severity is described in the companion article. These antibodies should prove useful in studying the role of oxidatively modified lipoproteins as well as other oxidatively modified proteins in atherogenesis.

Reliability and validity of six systems for the clinical assessment of disease activity in systemic lupus erythematosus.

Six systems for defining and evaluating disease activity in patients with systemic lupus erythematosus (SLE) (the Ropes system, the National Institutes of Health [NIH] system, the New York Hospital for Special Surgery system, the British Isles Lupus Assessment Group [BILAG] scale, the University of Toronto SLE Disease Activity Index [SLE-DAI], and the Systemic Lupus Activity Measure [SLAM]) were tested on 25 SLE patients who were selected to represent a range of disease activity. The patients were evaluated independently by 2 physicians on 2 occasions approximately 1 month apart. Differences between patients demonstrated the largest source of variation in scores, accounting for 56-84% of the total variance, depending on the instrument. Differences between physicians (i.e., error) showed the next largest variation, 11-28% of the total variance, and differences between visits made up 5-16% of the total. The BILAG, SLE-DAI, and SLAM had the best inter-visit and inter-rater reliability. Convergent validity was shown by the strong correlations of scores among the different instruments (r = 0.81-0.97). All instruments correlated highly with the physicians' clinical impression of disease but less well with their evaluation of disease severity. The number of American Rheumatism Association criteria for SLE that were met by the patients correlated poorly with the physicians' global evaluation and with the scores of the instruments. The patients' self-reported disease activity scores correlated highly with the physicians' assessments of disease activity (r = 0.85-0.91), and the mean values from self-reports and from physicians' assessments were nearly equal. In contrast, severity scores correlated less well between self-reports and physician assessments (r = 0.49-0.69), and mean self-reported severity values were lower than the means from physicians. The BILAG, SLE-DAI, and SLAM systems appear to have better psychometric properties than the others for clinical research.

Interleukin-1, endotoxin or tumor necrosis factor/cachectin enhance the level of plasminogen activator inhibitor messenger RNA in bovine aortic endothelial cells.

It is known that either endotoxin (LPS) or interleukin-1 (IL-1) increase the activity of plasminogen activator inhibitor (PAI) in the culture media of human and bovine endothelial cells. We have confirmed these results in bovine aortic endothelial cells (BAEC). To determine if this effect was mediated by increases in the level of PAI messenger RNA (mRNA) we examined the effects of these cytokines on PAI mRNA levels in BAEC, using RNA blot analyses. Treatment of BAEC with either IL-1, LPS, or human recombinant tumor necrosis factor/cachectin (TNF) dramatically increased the level of PAI mRNA. Since elevated levels of PAI will decrease fibrinolytic potential, this mechanism is in concert with the known increase in in vivo procoagulant potential induced by these agents and could contribute to thromboembolic phenomena.

Low density lipoprotein undergoes oxidative modification in vivo.

It has been proposed that low density lipoprotein (LDL) must undergo oxidative modification before it can give rise to foam cells, the key component of the fatty streak lesion of atherosclerosis. Oxidation of LDL probably generates a broad spectrum of conjugates between fragments of oxidized fatty acids and apolipoprotein B. We now present three mutually supportive lines of evidence for oxidation of LDL in vivo: (i) Antibodies against oxidized LDL, malondialdehyde-lysine, or 4-hydroxynonenal-lysine recognize materials in the atherosclerotic lesions of LDL receptor-deficient rabbits; (ii) LDL gently extracted from lesions of these rabbits is recognized by an antiserum against malondialdehyde-conjugated LDL; (iii) autoantibodies against malondialdehyde-LDL (titers from 512 to greater than 4096) can be demonstrated in rabbit and human sera.

Recombinant human tumor necrosis factor induces acute reductions in food intake and body weight in mice.

We examined the effects of treatment with rHuTNF on food consumption and body weight in C3H/HeJ mice. rHuTNF was administered intraperitoneally either by injections of 3, 12, or 24 micrograms twice a day or by implantation of osmotic pumps that released 0.75, 3, or 12 micrograms per day. Dose-dependent reductions in both food intake and weight were induced by rHuTNF. However, in spite of continued exposure to rHuTNF, the mice developed a resistance to rHuTNF and resumed their pretreatment food intake and weight. Non-immunological factors may play a role in the development of this tolerance, since it developed rapidly and faded within 2 wk of cessation of exposure to rHuTNF.

Tumor necrosis factor not detectable in patients with clinical cancer cachexia.

Tumor necrosis factor (TNF) has been implicated in the pathogenesis of cachexia in neoplastic and infectious diseases. To assess the relationship between TNF and weight loss among cancer patients, we assayed TNF levels in serum from 19 patients who had lost 8%-40% of premorbid weight. The weight loss experienced by these patients was not attributable to anticancer therapy, gastrointestinal disorders, or other medical problems. TNF was measured using a quantitative sandwich enzyme-linked immunosorbent assay that is sensitive to human TNF in serum at concentrations greater than or equal to 40 pg/mL. No TNF was detected in serum samples from the 19 patients studied.

Antibodies against amino acids 1-15 of tumor necrosis factor block its binding to cell-surface receptor.

Human tumor necrosis factor (hTNF) mediates a variety of biologic activities, which are dependent on the attachment of hTNF to cell-surface receptors. To identify regions of the hTNF protein involved in binding hTNF to its receptor, we prepared five synthetic peptides [hTNF-(1-15), hTNF-(1-31), hTNF-(65-79), hTNF-(98-111), and hTNF-(124-141)] and two hydroxylamine cleavage fragments [hTNF-(1-39) and hTNF-(40-157)] of hTNF. The hTNF-synthetic peptides and hTNF fragments were tested in hTNF receptor binding assays and in two biologic assays: cytolysis of tumor cells and suppression of lipoprotein lipase in adipocytes. Neither the synthetic peptides nor hTNF fragments were active agonists or antagonists in these assays. The synthetic peptides were also conjugated to thyroglobulin, and peptide-specific antisera were raised. All five peptide-thyroglobulin conjugates induced antibody responses to the immunizing peptide and to hTNF. Each antiserum was tested for antagonist activity in hTNF binding assays. Only antisera raised against hTNF-(1-15) or hTNF-(1-31) and antisera against whole hTNF blocked binding. IgGs purified from these three antisera also block hTNF-induced cytolysis and lipoprotein lipase suppression. We conclude that antibodies that recognize the N-terminus of hTNF block the attachment of hTNF to its cellular receptor and inhibit the biologic effects of hTNF.

Tumors secreting human TNF/cachectin induce cachexia in mice.

Anorexia and weight loss are serious complications that adversely effect the prognosis of cancer patients. It has been suggested that TNF/cachectin may cause cachexia. To determine if TNF/cachectin can induce progressive weight loss in tumor-bearing animals, a clone of the human TNF/cachectin gene was isolated and inserted into a mammalian expression vector. This construct was transfected into CHO cells, and a cell line (CHO/TNF-20) that secretes TNF/cachectin was isolated. A cell line (CHO/CMV-Neo) that contains the same expression vector without the TNF/cachectin gene was also isolated. Nude mice injected intraperitoneally with CHO/TNF-20 cells died more quickly than mice injected with CHO/CMV-Neo cells. Eighty-seven percent of mice inoculated intramuscularly with CHO/TNF-20 cells developed severe cachexia and weight loss. All mice bearing CHO/CMV-Neo tumors maintained or increased their body weight. We conclude that mice bearing tumors that secrete TNF/cachectin develop progressive wasting and die more quickly than mice bearing control tumors.

Mutant ras-encoded proteins with altered nucleotide binding exert dominant biological effects.

We report that residues Lys-16 and Asp-119 play critical roles in the guanine nucleotide binding and, consequently, the biological function of the Ha-ras-encoded protein (Ha). Substitution of an asparagine residue for Lys-16 reduces the affinity of Ha for GDP and GTP by a factor of 100 but does not alter the specificity of nucleotide binding. The replacement of Asp-119 with an alanine residue reduces the affinity of Ha for GDP and GTP by a factor of 20 and reduces the relative affinity of Ha for GDP over IDP from 200-500 to 10. Based on these observations, a structural model for the GDP/GTP-binding site of Ha is proposed. By microinjecting purified proteins into NIH 3T3 cells, we observed that the ability of [Ala119]Ha to induce changes characteristic of cellular transformation was much greater than that of normal Ha and similar to that of the oncogenic [Val12, Thr59]Ha. In this assay, [Asn16]Ha and [Val12, Asn16, Thr59]Ha were similar in potency to normal Ha. In yeast cells, Ha proteins with reduced nucleotide affinity exert a dominant temperature-dependent lethality that is avoided by the coexpression of the activated yeast ras gene [Ala18, Val19]RAS2. We interpret the biological consequences of reducing the nucleotide affinity of ras proteins in terms of two opposing factors: a growth-promoting effect, resulting from an increase in the GDP-GTP exchange rate, and a growth-limiting effect, resulting from an increase in the nucleotide-free ras protein species.

Dysdifferentiative nature of aging: age-dependent expression of mouse mammary tumor virus and casein genes in brain and liver tissues of the C57BL/6J mouse strain.

Many aspects of the aging process could be the result of cells slowly drifting away from their proper state of differentiation. This possibility has been studied by searching for an age-dependent increase in the expression of specific genes in tissues where expression of these genes would not normally be expected. In these studies, cDNA probes of specific genes are used in a DNA X NA hybridization assay to detect possible complementary RNA sequences in tissues of different-aged animals. Using this technique in past experiments, a qualitative increase in the RNA sequence complexity of mouse leukemia virus (MuLV) and a quantitative increase in the amount of alpha- and beta-globin RNA were found with increasing age in the brain and liver of the C57BL/6J mouse strain. We report here a similar age-dependent qualitative increase in the RNA sequence complexity for mouse mammary tumor virus (MMTV) but no quantitative or qualitative age-dependent change in casein RNA sequences for the same tissues and mouse strain.

Quantification of deposition of neutrophilic granulocytes on vascular grafts in dogs with 111In-labeled granulocytes.

A new radioisotopic technique has been developed for quantification of deposition of neutrophilic granulocytes on vascular grafts. Nine healthy mongrel dogs underwent bilateral femoral artery resection and reconstruction with grafts of femoral vein and Gore-Tex. Pure granulocytes that had been separated from whole blood by centrifugal elutriation were labeled with 111In-tropolone in plasma. The granulocyte harvesting efficiency was 25 +/- 12%, and the labeling efficiency was 87 +/- 7%. Three hours after injection of labeled granulocytes and 2 hours after reperfusion, the grafts were harvested and cut into several segments for study of areas of anastomoses and midsections. On the basis of the radioactivity in the blood and in anastomotic and graft sections, the area of graft sections, and the neutrophilic granulocyte and differential leukocyte counts, the number of neutrophilic granulocytes adherent to a unit area and the total number of neutrophilic granulocytes on graft sections were calculated. These quantifications of the deposition of neutrophilic granulocytes indicated that the midsections of Gore-Tex grafts retained more neutrophilic granulocytes than did the midsections of vein grafts. Although the anastomotic areas retained more neutrophilic granulocytes than did the midsections of vein grafts, the opposite finding prevailed for the Gore-Tex grafts. A major fraction of neutrophilic granulocytes on Gore-Tex grafts was incorporated into thrombus. Semiquantitative information obtained by scintigraphy of the deposition of neutrophilic granulocytes on vascular grafts also confirmed this observation.

Partial expression of endogenous mouse mammary tumor virus in mammary tumors induced in BALB/c mice by chemical, hormonal, and physical agents.

The possible interaction of environmental factors with the endogenous mouse mammary tumor virus (MMTV) genome in the development of mammary tumors in the low-tumor-incidence BALB/c mouse strain was examined. Tumors were induced in virgin female animals by treatment with chemical carcinogen 7,12- dimethylbenz[alpha]anthracene or urethan, with or without prolonged hormonal stimulation, or by X-irradiation. Concomitant hormonal stimulation resulted in increased tumor incidences compared with those induced by chemical carcinogen treatment alone. The frequency of tumor induction by irradiation alone or in combination with urethan or prolactin stimulation was very low. MMTV expression in the mammary tumors was assayed by nucleic acid hybridization and by immunohistochemical staining. Depending upon the treatment group, 0 to 89% of the tumors contained detectable levels of MMTV RNA (>/=0.0005% of the total cellular RNA). Tumors which contained detectable viral transcripts exhibited only low levels of MMTV RNA, which did not appear to represent the accumulation of RNA sequences homologous to the entire MMTV genome; synthesis of MMTV structural proteins was detected in only one tumor. Viral RNA-positive tumors were generally associated with a longer latent period. MMTV RNA expression occurred in tumors classified histologically as adenoacanthomas, as well as in mammary adenocarcinomas, although the cell types in the adenoacanthomas expressing viral RNA were not identified. It does not appear that expression of the endogenous MMTV genome is required for maintenance of all mammary tumors in BALB/c mice, although partial genome expression undetectable by the methods employed cannot be ruled out. Linear regression analyses were performed. The mean time to tumor appearance and the percentage of tumors which were MMTV RNA positive were found to vary linearly as a function of the total dose of 7,12-dimethylbenz[alpha]anthracene administered. The percentage of tumors which were MMTV RNA positive was also shown to be linearly related to the mean time to tumor appearance. These relationships provide a basis for predictions in the BALB/c system related to these parameters.

Hormonal regulation of murine mammary tumor virus RNA expression during mammary tumorigenesis in BALB/c mice.

The effects of glucocorticoids and prolactin on murine mammary tumor virus (MuMTV) RNA expression in preneoplastic outgrowth lines and mammary tumors in BALB/c mice were investigated. Hyperplastic alveolar nodules (HAN) and a ductal hyperplasia (DH) are induced in virgin BALB/c mice by prolonged hormonal stimulation or treatment with 7,12-dimethylbenz(a)anthracene or both. Mice bearing HAN or DH outgrowth lines and mammary tumors that arose from the outgrowth lines were treated with glucocorticoids or prolactin. MuMTV RNA was quantitated by hybridization with a representative complementary DNA probe specific for MuMTV RNA. Prolactin treatment did not increase MuMTV RNA in the BALB/c HAN or DH outgrowth lines or tumors. MuMTV RNA increased after glucocorticoid treatment in the C3, C4, and C5 HAN outgrowth lines and in tumors that arose from the D1, D2, C4, and C5 HAN and CD8 DH outgrowth lines. No increase in MuMTV RNA with glucocorticoid treatment was observed in the D1 or D2 HAN outgrowth line, in the CD8 DH outgrowth lines, and in tumors that arose from the C3 HAN outgrowth line. The ability of glucocorticoids to stimulate MuMTV expression was specific since the response was dose dependent and specific for glucocorticoid hormones. Glucocorticoid treatment did not increase the level of type C viral RNA in the majority of hormone- or 7,12-dimethylbenz(a)anthracene-induced HAN outgrowth lines or tumors. These observations suggested that glucocorticoids may influence MuMTV expression during mammary tumorigenesis in BALB/c mice.