Association of simian immunodeficiency virus Nef with cellular serine/threonine kinases is dispensable for the development of AIDS in rhesus macaques.

The nef gene of simian immunodeficiency virus (SIV) is essential for high viral load and induction of AIDS in rhesus monkeys. A mutant form of the SIVmac239 Nef, which contains changes in a putative SH3-binding domain (amino acids 104 and 107 have been changed from PxxP to AxxA), does not associate with cellular serine/threonine kinases, but is fully active in CD4 downregulation and associates with the cellular tyrosine kinase Src. Infection of two rhesus macaques with SIVmac239 containing the mutant AxxA-Nef caused AIDS and rapid death in both animals. No reversions were observed in the majority of nef sequences analyzed from different time points during infection and from lymphatic tissues at the time of death. Our findings indicate that the putative SH3-ligand domain in SIVmac Nef and the association with cellular serine/threonine kinases are not important for efficient replication and pathogenicity of SIVmac in rhesus macaques.

Independence of herpesvirus-induced T cell lymphoma from viral cyclin D homologue.

Cyclin D family members are cellular protooncogenes, and their viral homologues in the Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus type 8 [HHV-8]) and the closely related Herpesvirus saimiri have been implicated as putative cofactors of viral transformation and pathogenesis. KSHV is regularly found in Kaposi's sarcoma and in the primary effusion B cell lymphoma and Castleman's disease associated with immunosuppression and AIDS. H. saimiri strain C488 transforms human and marmoset T cells in vitro and causes polyclonal T cell lymphoma in New World monkeys. The viral cyclins stimulate cell cycle progression of quiescent fibroblasts, and they form active cyclin-dependent kinase (CDK)6 complexes of broad substrate specificity that can resist and downregulate cellular CDK inhibitors. This study shows that the viral cyclin of H. saimiri strain C488 is not required for viral replication, T cell transformation, and pathogenicity in New World primates.

Complete withdrawal of immunosuppression in kidney allograft recipients: a prospective study in rhesus monkeys.

BACKGROUND: We previously reported the successful withdrawal of immunosuppression in kidney-allografted rhesus monkeys. Recipients had received pretransplant blood transfusions and cyclosporine (CsA) immunosuppression for 6 to 12 months. One animal is still alive at more than 15 years after transplantation. Our hypothesis was that the sharing of a single DR antigen between blood donor and recipient, and the sharing of the same DR antigen with the kidney donor, may be beneficial to allograft survival. We now report on the results from a prospective study. METHODS: The animals received three pretransplant blood transfusions from a single donor sharing one DR antigen with the recipient. Subsequently, a life-supporting kidney from a donor sharing the same DR antigen was transplanted. CsA was given for at least 6 months after transplantation. RESULTS: Two animals rejected their graft at 5-8 weeks after cessation of CsA treatment. One animal is still alive at 700 days after transplantation. This animal showed MLR nonreactivity to its kidney donor, similar to the animal at more than 15 years after transplantation. CONCLUSION: These results demonstrate that withdrawal of immunosuppression may be a realistic option in kidney graft patients under careful immunological monitoring of donor-specific immunity.

Prevention of renal allograft rejection in primates by blocking the B7/CD28 pathway.

BACKGROUND: There is accumulating evidence that blockade of the costimulatory pathways offers a valid approach for immune suppression after solid organ transplantation. In this study, the efficacy of anti-CD80 and anti-CD86 monoclonal antibodies (mAbs) in combination with cyclosporine (CsA) to prevent renal allograft rejection was tested in non-human primates. METHODS: Rhesus monkeys were transplanted with a partly major histocompatibility complex-matched kidney on day 0. Anti-CD80 and anti-CD86 mAbs were administered intravenously daily for 14 days starting at day - 1. CsA was given intramuscularly for 35 days starting just after transplantation. The kidney function was monitored by determining serum creatinine levels. RESULTS: The combination of anti-CD80 and anti-CD86 mAbs completely abrogated the mixed lymphocyte reaction. Untreated rhesus monkeys rejected the kidney allograft in 5-7 days. Treatment with anti-CD80 plus anti-CD86 mAbs resulted in a significantly prolonged graft survival of 28+ 7 days (P=0.025). There were no clinical signs of side effects or rejection during treatment. Kidney graft rejection started after the antibody therapy was stopped. The anti-mouse antibody response was delayed from day 10 to 30 after the first injection. No difference in graft survival was observed between animals treated with CsA alone or in combination with anti-CD80 and anti-CD86 mAbs. However, treatment with anti-CD80 and anti-CD86 mAbs reduced development of vascular rejection. CONCLUSIONS: In combination, anti-CD80 and antiCD86 mAbs abrogate T-cell proliferation in vitro, delay the anti-mouse antibody response in vivo, and prevent graft rejection and development of graft vascular disease in a preclinical vascularized transplant model in non-human primates.

Mortality after coronary angioplasty and coronary artery bypass surgery (the national Medicare experience).

Mortality rates for Medicare patients who underwent coronary artery bypass surgery were compared with those who had angioplasty or angioplasty and bypass surgery. Two data sets were used for this study: The first contained information on demographic factors, co-morbidities and subsequent mortality on all 96,666 Medicare patients who had bypass surgery or angioplasty in 1985; the second contained additional detailed clinical data collected using the MedisGroups method on a random sample of 2,931 revascularization patients from 6 states. From the national data set 30-day and 1-year mortality rates were 3.8 and 8.2% for 25,423 angioplasty patients and 6.4 and 11.8% for 71,243 bypass surgery patients (p less than 0.001 for both time periods). Mortality rates for the MedisGroups data were 4.4 and 8.5% for the angioplasty patients and 6.5 and 11.9% for the bypass surgery patients. After eliminating patients admitted with a myocardial infarction, mortality rates were 1.9 and 6.0% for 632 angioplasty patients and 5.1 and 10.8% for 1,730 bypass surgery patients. The risk-adjusted relative risk of mortality for bypass surgery versus angioplasty was 1.72 (p = 0.001) for all patients, 2.15 (p less than 0.001) for low-risk patients and 0.90 (p = not significant) for high-risk patients. Results suggest that low-risk patients have better survival with angioplasty because of lower short-term mortality.

Live attenuated SIV vaccines are not effective in a postexposure vaccination model.

Live attenuated simian immunodeficiency virus (SIV) vaccines, like nef deletion mutants, have been the most effective vaccines tested in the SIV/macaque model so far. The efficacy of live attenuated SIV vaccines in therapeutic vaccination and postexposure prophylaxis has not been determined. Inoculation of macaques with a pathogenic challenge virus and an attenuated SIV vaccine at the same time mimics postexposure vaccination, whereby vaccination with the attenuated virus is performed as rapidly as possible after exposure to pathogenic SIV. In the study presented here, four rhesus macaques were coinfected with pathogenic SIV and a nearly 3000-fold excess of a nef deletion mutant of SIV. Four macaques received pathogenic SIV and an approximately 200-fold excess of a nef deletion mutant expressing interleukin 2 (IL-2). The IL-2-expressing SIV had been previously constructed to enhance the immunogenicity of live attenuated SIV vaccines. All coinfected macaques had a high viral load, and some of them developed AIDS-like symptoms and pathological alterations rapidly. In the presence of pathogenic SIV, both live attenuated SIV vaccines did not protect from disease in this postexposure vaccination model.

Cytokine gene transcription in simian immunodeficiency virus and human immunodeficiency virus-associated non-Hodgkin lymphomas.

Infection of rhesus monkeys with SIV leads to AIDS-like symptoms. Similar to human AIDS patients, some monkeys develop B cell non-Hodgkin lymphoma (NHL). We determined transcription of cytokine genes regulating the activation of B and T cells, which play a role in intratumoral immune surveillance. Therefore, we compared the transcription of the cytokine genes encoding IL-2, IL-4, IL-6, IL-10, IFN-gamma, TNF-alpha, and TGF-beta1, and the Epstein-Barr virus-encoded BCRF 1 gene, in cells from five monkey and two human tumor specimens. The immune-suppressive IL-10 and TGF-beta1 genes were predominantly transcribed in all tumor specimens analyzed. Cytokine gene transcription patterns appeared to be similar in human and animal tumor cells. The transcription patterns corresponded to their histological classification as diffuse large-cell lymphoma according to the REAL classification and as immunoblastic or centroblastic tumors according to the Kiel classification. The determination of cytokine gene transcription pattern in the NHL may improve our understanding of pathogenesis and immune surveillance in this heterogeneous group of tumors. Our data show that SIV-associated NHLs of rhesus monkeys are comparable to human HIV-1-associated EBV-positive non-Hodgkin lymphoma.

Amitosis and endocycles in early cultured mouse trophoblast.

The possible occurrence of amitosis has been studied in nuclei of trophoblast outgrowths of mouse embryos placed in culture at the two-cell stage. By day 7 of culture, the inner cell mass has usually floated away, while the trophoblast outgrowth remains attached. Of 591 trophoblast cells from 25 embryos, 469 were uninucleate, 87 binucleate, 4 trinucleate, and in 31 the nuclei were attached to each other. In our interpretation, these come about through a process in which the nucleus stretches, then the nuclear membrane invaginates and finally constricts the nucleus into two parts. The resulting nuclei, asymmetric in size and in amount and arrangement of heterochromatin and nucleoli, lie side-by-side. We conclude that these cells with two or more attached or separate nuclei represent stages in true amitosis. In Table 1, amitosis is compared with mitosis without cytokinesis and with cell fusion, both of which can also give rise to binucleate and multinucleate cells. Mitosis without cytokinesis does not agree in any respect with the characteristics of amitosis, whereas at least a few similarities exist between cell fusion and amitosis. However, amitosis may give rise to near-haploid nuclei, which cannot be produced by mitosis or cell fusion. Simultaneously with amitosis, the nuclei grow through endocycles. In many nuclei, the heterochromatin is clearly underreplicated, while the nucleoli are numerous and often of enormous size, probably reflecting amplification of the rRNA genes.

Relaxing retinotomies and retinectomies. Surgical results and predictors of visual outcome.

Functional and anatomic success after relaxing retinotomy may be limited by recurrent retinal detachment or severe hypotony. Fifty-four consecutive eyes undergoing relaxing retinotomy for proliferative vitreoretinopathy (42 eyes) and trauma (12 eyes) were analyzed to determine whether perioperative factors, including size and location of the retinotomy, influenced visual or anatomic outcome. After 6 months' minimum follow-up, anatomic success (retina attached posterior to buckle and an intraocular pressure of 3 mm Hg or more) was achieved in 35 eyes (64%). Functional success (visual acuity of 5/200 or better) was achieved in 14 eyes (26%). Factors predicting functional success by stepwise logistic regression analysis included a preoperative visual acuity of hand motions or better and location of the retinotomy in the superior four clock hours of the fundus. Causes of anatomic failure included proliferative vitreoretinopathy (11 eyes) and severe hypotony or phthisis (8 eyes). Superior location of the retinotomy and visual acuity of hand motions or better favorably influenced visual outcome after relaxing retinotomy.

Successful suppression of the early rejection of pig islets in monkeys.

Primary nonfunction (PNF) is seen very frequently after xenogeneic transplantation of islets of Langerhans. In a pig-to-rat model we recently observed that no PNF occurs when the islets are kept in culture at 37 degrees C for 1-2 weeks prior to transplantation. In order to investigate the rejection mechanisms in a preclinical model, we transplanted cultured porcine islets under the capsule of both kidneys in four cynomolgous monkeys. Islets were isolated from adult sows by means of digestion with Liberase in University of Wisconsin solution (UWS). The digest was purified by a density gradient of OptiPrep in UWS. Highly purified (>95%) islets were cultured 1-2 weeks in RPMI. All monkeys showed significant titers of preformed anti-pig antibodies. The immunosuppression of the monkeys consisted of cyclophosphamide (Cy) (2 days), cyclosporin A (CsA), and prednisolone. Anticipating a fast rejection we carried out nephrectomies at different time points within 2 weeks after transplantation. Following unilateral nephrectomy, well-preserved islets with no signs of rejection were observed between 3 and 7 days posttransplant. Later, between days 11 and 15 posttransplant, histology in the first three animals demonstrated no islets. In the fourth monkey histology on day 11 showed islets with excellent morphology and some small focal infiltrates. The highest CsA blood levels (around 1000 ng/ml) were found in animals with the best graft survival. We conclude that cultured porcine islets can be grafted without hyperacute rejection in monkeys with preformed anti-pig antibodies. In the presence of high levels of CsA only marginal signs of a cellular immune response were observed 11 days after transplantation.

BUN as a risk factor for mortality after coronary artery bypass grafting.

BACKGROUND: Although information on blood urea nitrogen (BUN) is universally available for patients who undergo coronary artery bypass grafting, BUN has not often been considered as a risk factor for mortality. This study assessed BUN as a risk factor for CABG patients. METHODS: Four data sets were evaluated that differed with respect to the types of patients and available patient information. In each of these data sets logistic regression analysis was used to examine the relationship between BUN and mortality after adjusting for other risk factors. RESULTS: Blood urea nitrogen level was strongly associated with mortality in each of the data sets. After adjustment for the available risk factors other than creatinine level, patients with BUN levels greater than 30 mg/dL had a relative odds of mortality ranging between 1.86 and 2.49 (p < 0.0001 in three of the data sets). Even after adjustment for creatinine level as well as the other variables, BUN was statistically significant at the p less than 0.01 level for three of the data sets. CONCLUSIONS: The results suggest that BUN provides additional information on cardiac function that supplements the information provided by other risk factors.

Incidence and origin of symmetric and asymmetric dicentrics in Bloom's syndrome.

The incidence and origin of dicentric chromosomes has been analyzed in Bloom's syndrome (BS) lymphocytes. In diploid cells, the dicentrics are predominantly asymmetric, consisting of two nonhomologous chromosomes. In contrast, in tetraploid cells, the majority of dicentrics are formed by two homologs, which, judging from Q-banded metaphases, have broken at identical points. Our earlier assumption that most, possibly all, chromosome abnormalities in BS originate in S-G2 is further supported by the present observation that only 26/79 cells with dicentrics also had a fragment. In other words, the dicentrics have not arisen in the previous G1 but in S-G2 of a preceding cell cycle. The symmetric dicentrics would arise in diploid cells from an adjacent counterpart to a mitotic chiasma between two homologs, and in tetraploid cells, from an adjacent exchange between two sister chromosomes in a diplochromosome. The asymmetric dicentrics can be assumed to result primarily from segregation of an adjacent QR between two nonhomologous chromosomes. Based on the frequency of symmetric dicentrics in tetraploid cells (25/61 cells), and assuming that the ratio of adjacent exchanges to mitotic chiasmata is around 1/50, we predict that the chiasma frequency in diplochromosomes of BS lymphocytes will turn out to be as high as 20/cell.

Cytogenetics of Bloom's syndrome.

The quantitative aspects of Bloom's syndrome cytogenetics are reviewed. The most characteristic feature is an increased rate of homologous chromatid exchange, both sister chromatid exchange and mitotic crossing-over. Other phenomena are a tendency of somatic cells to fuse, an increased rate of chromosome breaks, often with sister chromatid reunion, formation of nonhomologous quadriradials, and occurrence of allocyclic and triradial chromosomes. Mitotic chiasmata are situated highly nonrandomly, preferably in Q-dark regions. Chromosomes containing chiasma "hot-spots" appear to contain more active genes than similarly sized control chromosomes. They also contain a high proportion of localized oncogenes. Bloom's syndrome homozygotes show a high incidence of cancer (1/4). This may depend on a) the high rate of homozygosity resulting from mitotic crossing-over, which would allow the expression of recessive cancer genes; b) unequal crossing-over would amplify these genes; c) chromosome structural changes that might transfer oncogenes to new locations and, thus, activate them; and d) immunodeficiency, which would promote malignant growth.

The behavior of heterochromatin in mouse and human nuclei.

The arrangement of heterochromatin in various human and mouse nuclei has been analyzed with C banding. In most nuclei of 7-day mouse trophoblast, the heterochromatin consists of twin dots, or bigger clumps, apparently attached to the nuclear membrane. This finding agrees with the observation that most of these nuclei, which range from diploid to highly polyploid, show endomitotic stages. No polarization of heterochromatin in a Rabl orientation is seen in the trophoblast nuclei. Neither is a Rabl orientation found in the interphases of cultured human lymphocytes or fibroblasts. From their telophase arrangement, the chromosomes have obviously spread rapidly around the nuclear membrane. In many of the giant mouse trophoblast cells in vivo and in vitro, heterochromatin is apparently underreplicated. The same is true of giant cells in human hydatidiform moles and cervical cancer. Of the 82 cervical cancers analyzed, 46 showed chromocenters, and each tumor was characterized by its own pattern of heterochromatin.

The course of endomitosis in human cells.

The course of endomitosis in human hydatidiform moles has been analyzed. It differs from the classical description of endomitosis in that endoprophase is completely missing and, very probably, so is a typical interphase. The chromosomes are even less synchronized in their replication and condensation cycle than in normal mitosis. At no point do all chromosomes decondense, but a part remains condensed while others are extended and in the process of synthesizing DNA. Even two paired sister chromosomes may replicate nonsynchronously. The latest replicating chromosome is usually a large darkly staining chromosome, which we tentatively identify as the inactive X. No DNA synthesis takes place during "endometaphase" or "endoanaphase" stages, when the chromosomes are most condensed. Some polyploid "endoanaphases" or "endotelophases" with stretched out chromosomes obviously represent end-stages of the endomitotic pathway, and the nuclei are in the process of reverting into evenly stained nuclei. In some "endometaphases," a near-haploid number of chromosomes can be counted. In others, the endochromosomes seem to be compound structures consisting of several chromosomes that have not separated during the previous endomitoses. This is seen also in normal trophoblast and cervical cancer. In large cancer cells, such bundles can be seen in the process of falling into individual chromosomes.

Correlation of rates of coronary artery bypass surgery, angioplasty, and cardiac catheterization in 305 large communities for persons age 65 and older.

OBJECTIVE: The rate of coronary artery bypass surgery (CABG) has been shown to vary greatly across geographic regions. This study examined whether these rates were associated with the rate of coronary artery angioplasty (PTCA) and with other community characteristics. DATA SOURCES/STUDY SETTING: The health care financing administration provided the number of Medicare hospitalizations in 1988 for conditions and procedures related to coronary artery disease. Information on physicians and hospitals was obtained from the Area Resource File, and the number of persons in each age, sex, and race category was obtained from US. census data. STATISTICAL METHODS: Age-and sex-adjusted hospitalization rates based on the patient's zip code of residence were calculated at the level of the Metropolitan Statistical Area (MSA) for white patients age 65 or older. Rates were obtained for 305 MSAs for CABG, PTCA, cardiac catheterization, angina, and myocardial infarction. PRINCIPAL FINDINGS: The rate of cardiac catheterization had a correlation of .72 with the CABG rate and .64 with the PTCA rate. The correlation of the PTCA and CABG rates with each other was .49. This correlation was not charged by adjusting for the rates of hospitalization for angina or myocardial infection, but it was reduced to only .05 (ns) after adjusting for the rate of cardiac catheterization. The rates of all three procedures had rank correlations of about .15 with the density of thoracic surgeons and about .30 with the density of hospitals with cardiac catheterization and open heart surgery units. CONCLUSIONS: Community CABG and PTCA rates tend to move in the same direction due to community factors that also affect the rates of cardiac catheterization. These community factors do not appear to include the rate of coronary artery disease, but may include resources or attitudes toward aggressive treatment of coronary artery disease.

The relationship between adjusted hospital mortality and the results of peer review.

This study assessed the relationship between the Health Care Financing Administration adjusted mortality rate for a hospital and the errors in care found by the peer review process. The three data sets used were: (1) the 1987-1988 completed reviews from 38 peer review organizations (PROs) of 4,132 hospitals and 2,035,128 patients; (2) all 1987 hospital mortality rates for Medicare patients as adjusted by HCFA for patient mix; and (3) the 1986 American Hospital Association Survey. The PRO data were used to compute the percentage of cases reviewed from each hospital confirmed by a reviewing physician to have a quality problem. The average percentage of confirmed problems was 3.73 percent with state rates ranging from 0.03 percent to 38.5 percent. The average within-state correlation between the problem rate and the adjusted mortality rate for all PROs was .19 (p < .0001), but the correlations were much higher for relatively homogeneous groups of hospitals, .42 for public hospitals and .36 for hospitals in large metropolitan statistical areas (MSAs). These results suggest that the HCFA adjusted hospital mortality rate and the PRO-confirmed problem rate are related methods to compare hospitals on the basis of quality of care. Both methods may compare quality better if used within a group of homogenous hospitals.

Evaluation of the HCFA model for the analysis of mortality following hospitalization.

From 1987 through 1990, the Health Care Financing Administration (HCFA) evaluated variations in the mortality rates experienced by patients admitted to hospitals participating in the Medicare program. This study was conducted to evaluate the adequacy of the model used for that purpose. Detailed clinical data were gathered on 42,773 patients admitted to 84 statistically selected hospitals. The effect of risk adjustment using the HCFA model, which is based on claims data, was compared to a risk-adjustment model based on physiologic and clinical data. Models that include claims data were markedly superior to those containing only demographic characteristics in predicting the probability of patient death, and the addition of clinical data resulted in further improvement. The correlation of ranks of hospitals based on a model that uses only the claims data and on one that uses, in addition, clinical data, was .91. As a screen for the identification of "high (mortality) outlier" hospitals, the claims model had moderate sensitivity (81 percent) and specificity (79 percent), a high negative predictive value (90 percent), and a low positive predictive value (64 percent) when compared to the clinical model. The two mortality models gave similar results when used to determine which structural characteristics of hospitals were related to mortality rates: hospitals with a higher proportion of registered nurses or board-certified physician specialists, or with a greater level of access to high-technology equipment had lower risk-adjusted mortality rates. These data suggest that the current claims-based risk-adjustment procedure may satisfactorily be used to characterize variations in mortality rates associated with hospitalization. The procedure could also be used as a basis for further epidemiological analyses of factors that affect the probability of patient death. However, it does not positively identify outlier hospitals as providers of problematic care.

Age and gender patterns in motor vehicle crash injuries: importance of type of crash and occupant role.

To evaluate the interaction of gender, age, type of crash, and occupant role in motor vehicle crash injuries leading to hospitalization, we analyzed 1997 Wisconsin hospital discharge data for patients with primary E-code diagnoses of motor vehicle injuries. The overall ratio of males to females (M/F ratio) hospitalized for motor vehicle crash injuries was 1.33 (95% confidence interval (CI): 1.26-1.41). The M/F ratio varied by type of crash and differed for passengers and drivers. For injuries sustained in collisions between vehicles, the M/F ratio was 0.96 (95% CI: 0.87-1.05); in loss of control accidents the M/F ratio was 1.95 (95% CI: 1.76-2.17). Within each type of crash, the M/F ratio for drivers was similar to that for the entire type; the M/F ratio for passengers was about half of the type total. Expressed as rates of hospitalization per 100,000 people in the general population, hospitalizations of drivers in collisions with another motor vehicle increased steeply in males, but not in females, beginning at about age 70. For drivers in loss of control crashes, male rates exceeded female rates in all age groups, with peaks in the groups 15-24 and 85-89. For passengers, injury rates from collisions with other motor vehicles were greater for females, especially in the elderly, and injury rates from loss of control crashes were similar for both genders, with peaks at 15-24 and 85-94. The higher fatality of men in loss of control motor vehicle crashes, compared to women, suggests an important area for further investigation.

Platynosomum fastosum in ex-captive orangutans from Indonesia.

The liver fluke Platynosomum fastosum was identified upon necropsy of three ex-captive orangutans (Pongo pygmaeus) which had been part of a rehabilitation program for reintroduction to the wild. This trematode has not been reported in orangutans previously and is commonly found in cats in Southeast Asia. Cross infection from cats via intermediate hosts, to orangutans kept in captivity as pets, could explain their presence in the latter. Although P. fastosum caused intrahepatic and bile duct damage, death of the hosts could not be attributed solely to the presence of the liver fluke infection.