Platelet kinetics in dogs treated with a glycoprotein IIb/IIIa peptide antagonist.

Platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptor antagonists have been highly effective inhibitors of platelet aggregation in preclinical studies and in clinical trials. However, decreased platelet counts have been documented in preclinical studies and in some patients receiving GPIIb/IIIa antagonists. We evaluated changes in platelet kinetics and fate in dogs receiving the GPIIb/IIIa receptor antagonist RPR 109891 orally for 4 days. Dogs receiving RPR 109891 had a 22-52% decrease in platelet count with the nadirs at 3-5 days after initiation of treatment. Platelet survival time was reduced by 19%, and platelet half-life was reduced by 63%. Indium-111-labeled platelets were rapidly cleared from the blood within 1 hour after administration of RPR 109891 on treatment days 1 and 2. This clearing was associated with a sharp increase in radioactivity in spleen but not in liver or lung. Platelet clearance was markedly attenuated on treatment days 3 and 4. Platelet counts returned to baseline within 1 week after discontinuation of treatment. These data indicate that RPR 109891 causes rapid and selective sequestration of platelets in the spleen.

A spontaneously arising porcine model of bullous pemphigoid.

Bullous pemphigoid (BP) is an IgG-mediated autoimmune blistering disease targeting the hemidesmosomal proteins bullous pemphigoid antigens 1 and 2. Currently, there is no active animal model in which to dissect the immunopathogenic mechanism. We noticed that cutaneous blistering arose spontaneously in 12 adult Yucatan minipigs. Skin lesions consisted of turgid, isolated or clustered vesicles that occasionally evolved from erythematous and pruritic patches. Histopathological examination revealed subepidermal vesicles rich in intact and degranulated eosinophils. Antigen mapping and transmission electron microscopy confirmed that dermoepidermal separation took place in the lamina lucida of the epidermal basement membrane zone. Direct immunofluorescence revealed the presence of IgG deposited linearly at the dermoepidermal junction in seven of nine skin specimens examined. Indirect immunofluorescence testing confirmed the presence, in the serum from eight of eight affected pigs, of circulating basement membrane-specific IgG autoantibodies (titers 1 : 50 to 1 : 250). Using uncleaved and salt-split lip substrates, the autoantibodies were shown to target antigens situated not only at the basal, but also at the lateral and apical aspects of stratum basale keratinocytes. Immunoelectron microscopy confirmed that circulating IgG autoantibodies recognized hemidesmosomal antigen(s). ELISA, immunoblotting and immunoadsorption demonstrated that five of eight serum samples exhibited high immunoreactivity against BPAG2-NC16A peptides. This novel porcine acquired blistering dermatosis could be proposed as a valuable model to conduct immunomechanistic studies on the natural progression of BP, correlation of autoreactive T cells or autoantibodies with disease activity, and the role of eosinophils in the blistering process, as these diseases cannot be modeled easily in human patients or in murine passive transfer models.

Reduced bovine leukaemia virus proviral load in genetically resistant cattle.

The bovine leukaemia virus (BLV) is an exogenous retrovirus that is closely related to the human T cell leukaemia viruses. Genetic resistance and susceptibility to persistent lymphocytosis (PL), an advanced subclinical stage of infection characterized by a polyclonal expansion of the infected B cell population, have been mapped to structural motifs in bovine MHC DRB3 (class II) alleles. To determine whether alleles of DRB3 influence the number of BLV-infected B cells in peripheral blood, seven pairs of Holstein cows naturally infected with BLV were matched on the basis of DRB3 genotype (resistance or susceptibility to PL), age, and year of seroconversion. Flow cytometry was used to separate B cell populations that then were tested for the presence of provirus by a single-cell PCR methodology. Animals with the PL-resistance associated DRB3.2*11 allele had significantly fewer BLV-infected B cells than did age- and seroconversion-matched cows with DRB3 alleles associated with susceptibility to PL. Our results demonstrate that DRB3 or a closely linked gene may play a direct role in controlling the number of BLV-infected peripheral B cells in vivo. Association of MHC class II alleles with resistance to disease progression in cattle naturally infected with BLV provides a unique immunogenetic model for the study of host resistance to human and other animal retroviral infections.

Use of flow cytometry and RT-PCR for detecting gene expression by single cells.

We have developed a method for reliably detecting gene expression by individual, phenotypically defined cells. Cells were sorted by flow cytometry into 96-well plates containing a Nonidet P-40 (NP40)-based bysis solution. Reverse transcription (RT) of cellular major histocompatibility complex class II DQB and either bovine leukemia virus (BLV) env or tax/rex mRNA was subsequently conducted using gene-specific oligonucleotide primers. Two sequential rounds of PCR were then performed to co-amplify DQB and either BLV env or tax/rex cDNA. The PCR products were electrophoresed in 6% polyacrylamide gels and visualized by ethidium bromide staining. The BLV-infected BL3 cell line was used to establish the sensitivity of the method; cellular and viral mRNA were reproducibly detected in wells into which single BL3 cells were sorted. Additionally, BLV env mRNA from single infected cells was consistently detected in reactions containing as many as 1000 uninfected cells. By using this method, 0.012% +/- 0.002% of B cells from a BLV-infected cow with persistent lymphocytosis were found to express BLV tax/rex mRNA, whereas < or = 0.001% expressed BLV env mRNA. The combination of single-cell sorting and RT-PCR provides a powerful new tool to study viral transcription, host responses associated with progression of retroviral infections or other problems requiring determination of the frequency of cells expressing a particular gene(s).

The prevalence of proviral bovine leukemia virus in peripheral blood mononuclear cells at two subclinical stages of infection.

The bovine leukemia virus (BLV) is an oncogenic retrovirus that is associated with the development of persistent lymphocytosis (PL) and lymphoma in cattle. While B lymphocytes have been shown to be the primary cellular target of BLV, recent studies suggest that some T lymphocytes and monocytes may be infected by the virus. Because virally altered functions of monocytes and/or T cells could contribute to the development of lymphoproliferative disease, we sought to clarify the distribution of the BLV provirus in subpopulations of peripheral blood mononuclear cells in seropositive cows with and without PL. CD2+ T cells, monocytes, and CD5+ and CD5- B cells were sorted by flow cytometry and tested for the presence of BLV by single-cell PCR. We did not obtain convincing evidence that peripheral blood monocytes or T lymphocytes contain the BLV provirus in seropositive cows with or without PL. In seropositive cows without PL (n=14), BLV-infected CD5+ and CD5- B cells accounted for 9.2% +/- 19% and 0.1% +/- 1.8% of circulating B lymphocytes, respectively. In cows with PL (n=5), BLV-infected CD5+ and CD5- B cells accounted for 66% +/- 4.8% and 13.9% +/- 6.6% of circulating B lymphocytes, respectively. The increase in lymphocyte numbers in cows with PL was entirely attributable to the 45-fold and 99-fold expansions of infected CD5+ and CD5- B-cell populations, respectively. Our results demonstrate that B cells are the only mononuclear cells in peripheral blood that are significantly infected with BLV. On the basis of the absolute numbers of infected cells in seropositive, hematologically normal animals, there appear to be differences in susceptibility to viral spread in vivo that may be under the genetic control of the host.

Detection of bovine leukemia virus proviral DNA in individual cells.

We have developed a method of analyzing individual cells to detect proviral DNA of the bovine leukemia virus (BLV) using flow cytometry and PCR. Individual cells of the BL3* cell line, which contain multiple integrated copies of the BLV provirus, and the uninfected cell line BL3(0), were sorted into wells of a 96-well plate. Following cell lysis, portions of the BLV envelope (ENV) and cellular prolactin (PRL) genes were amplified simultaneously using PCR. Viral and cellular products of first-round PCR were amplified separately in a second round of PCR using "heminested" primers. Separation of the PCR products by polyacrylamide gel electrophoresis yielded distinct fragments of the predicted sizes. The operational sensitivity of this method for the detection of virus was > 90% when testing single infected cells. In addition, we were able to reliably amplify DNA from a single BL3* cell among as many as 10(5) BL3(0) cells and established that the sensitivity for detecting a single infected cell among 20, 100, or 1000 uninfected cells was at least 90%. Estimates of low percentages of infected cells were obtained by applying probability theory to results of experiments conducted on wells containing more than one cell. Using these methods, B lymphocytes obtained from the peripheral blood of BLV-infected cattle were tested for proviral DNA. BLV ENV was identified in 76.9 +/- 4.9% of single B cells tested from a seropositive animal with persistent lymphocytosis (PL), but in only 0.033 +/- 0.009% of B cells from another seropositive cow without PL.(ABSTRACT TRUNCATED AT 250 WORDS)

Mycobacterium bovis infection in a captive herd of Sika deer.

Infection with Mycobacterium bovis was diagnosed in a small privately owned herd of Sika deer. After postmortem examination of a deer with progressive pulmonary disease, diagnosis of infection with M bovis was confirmed by bacteriologic culture. The 2 remaining deer in this herd were euthanatized, necropsied, and confirmed to be infected with M bovis. Three cats in contact with the deer were also euthanatized and necropsied. One of these cats had lesions suggestive of mycobacterial infection in the colon and mesenteric lymph nodes. Infection of this cat with M bovis was not confirmed by bacterial culture. Mycobacteriosis, infrequently encountered in clinical veterinary practice, may be confused with disease caused by other infective agents or neoplasia. The zoonotic potential of these bacteria and a recent increase in human tuberculosis warrants continued surveillance of companion and food animal populations for mycobacterial infection.

Apparent retrovirus-induced immunosuppression in a yearling llama.

Immunosuppression suspected to be associated with retrovirus infection was diagnosed in an 18-month-old female llama. The llama had a 6-month history of weight loss, intermittent lameness, and infections that were nonresponsive to treatment. Serial CBC indicated persistent nonregenerative anemia and leukopenia characterized by absolute neutropenia and lymphopenia. Functional hypoplasia of myeloid and erythroid cell lines was detected in serial bone marrow biopsy specimens. Notable pathologic findings included inadequate hematopoiesis, generalized lymphoid hypoplasia and plasma cell depletion, and pulmonary alveolar histiocytosis. Pneumocystis carinii cysts and viral particles of the size and morphologic features consistent with the retrovirus family were observed in lung sections examined by transmission electron microscopy. Antemortem macrophage and postmortem lymph node cultures were positive for reverse transcriptase activity.

Moscow Hospital School.

Among the innovations introduced during the dynamic reign of Peter the Great in his attempt to bring Russia in line with countries in Western Europe was the foundation of the Moscow Hospital School in 1707. He entrusted the direction of the school to the Dutch physician and surgeon, Nicolas Bidloo, who dedicated himself to his students and to the organisation of the school and was an excellent teacher as well as an expert in anatomy. The large number of surgical instruments listed in a catalogue drawn up when the hospital was damaged by fire in 1738 testify to the quantity and variety of surgical procedures carried out at the hospital. Equal importance was given in the courses to theory and practice, and surgery was considered on a par with medicine at a time when surgeons in most European countries were considered to be an inferior category. The high standards of the school soon won it great renown and later medical schools in Russia were modeled on this first medical institution of higher education in the country.

Coronary care in New York City hospitals.

New York City, with six medical schools and an elaborate emergency medical system, would appear to be a locale with optimal resources for coronary care. To assess coronary care facilities and practices, a comprehensive questionnaire was prepared by a multidisciplinary committee and submitted to 72 hospitals. Seventy-nine percent (57/72) responded, including all medical school affiliated hospitals and 41 of the 45 with more than 300 beds. Twenty-nine percent (16/55) did not have a coronary care unit separate from a general intensive care unit. Thirty-three percent (18/55) of coronary care units were designed before 1970. Although 85% (46/54) of the coronary care units had a 1:2 registered nurse per patient ratio during days, only 43% (23/54) maintained this ratio at night. Central monitoring was available in all hospitals, but only 40% (21/52) indicated 24-hour continuous monitor observation. Forty-five percent (25/55) had cardiac catheterization facilities, and 43% (24/56) had an intra-aortic balloon available. Forty-six percent (26/57) of coronary care units transferred post-myocardial infarction patients to non-monitored general beds. Fifty-eight percent (31/53) offered dietary counseling to patients, and 33% (18/54) had an inpatient cardiac rehabilitation program. Thus, New York City hospitals demonstrate a marked variability in coronary care unit facilities and practices and a substantial number of hospitals deviate from published recommendations for coronary care units.

Blood gas stability and hematological changes in experimentally-induced acute porcine pleuropneumonia.

Blood gas and hematological responses to acute, mild Actinobacillus pleuropneumoniae infection of growing pigs was studied. Six pigs (average weight 10.1 kg) were experimentally infected intranasally with A. pleuropneumoniae serotype 5. Four pigs served as controls. Rectal temperatures and arterial blood for gas analysis and hematology were taken at 0, 8, 16, 24, 48 and 72 h postinfection. All infected pigs became febrile showing clinical signs typical of mild to moderate porcine pleuropneumonia; controls remained asymptomatic. Neutrophilia with bands and lymphopenia were observed only in infected pigs. Arterial partial pressures of O2 and CO2, and pH did not change in infected pigs. All pigs were killed after 72 h, and lungs were examined and cultured. Gross and microscopic lesions consistent with porcine pleuropneumonia were seen in 3/6 and 5/6 infected lungs, respectively. Control lungs were grossly normal with no histological evidence of pleuropneumonia. We conclude that in mild, acute porcine pleuropneumonia as established experimentally, a leukogram typical of acute inflammation and stress is seen; however, hypoxemia and alveolar hypoventilation are not features of this form of the disease.

Clinical practice of medical school faculties: an AAMC survey of problems and issues.

This study reports the results of a survey by the Association of American Medical Colleges (AAMC) to identify the salient problems and issues regarding medical school faculty clinical practice in the views of faculty members and administrators in academic medical centers. The population surveyed consisted of university vice-presidents for health affairs, teaching hospital directors, and medical school deans, department chairmen, and faculty representatives. A content analysis of the narrative responses revealed that the apportionment of faculty time to create the appropriate institutional balance among teaching, research, and service missions was the most frequently mentioned concern. Other dominant themes included threats to the patient base needed for teaching and research and/or to future income posed by the new competitive and reimbursement environment and changes required in modes of faculty practice to respond to the new environment. The latter included the need for organizing practice activities into a "true group practice," a change that would accommodate growing demands of purchasers for integrated systems of care. Hospital directors were particularly concerned with issues of faculty-hospital relationships. Results of the survey provide an agenda for a recently formed AAMC ad hoc Committee on Faculty Practice, which will propose initiatives by the AAMC to assist its member institutions in responding to the issues raised.

Soleus motoneurone excitability in man: an indirect approach for obtaining quantitative data.

Using combinations of H and T reflexes, spatial summation has been studied in the human soleus motoneurone pool. With certain assumptions, mathematical treatment of the results yielded estimates of the thresholds of motoneurones to monosynaptic activation by Ia nerve fibres. It was found that, on average, about 62 EPSPs were required to discharge a motoneurone and that a single Ia fibre supplied about 139 neurones.

Acute hemodynamic alterations after aortic valve replacement with the glutaraldehyde-treated porcine heterograft prosthesis.

Twenty-two patients underwent aortic valve replacement for acquired valvular heart disease with the porcine heterograft prosthesis. In the immediate postoperative period, a transient decrease in the cardiac index (CI) (preoperative 3.0 +/- 0.14 to 2.45 +/- 0.16 L. per minute per square meter, p less than 0.05) and an increase in the systemic vascular resistance (SVR) (preoperative 1,078 +/- 62 to 1,287 +/- 103) dynes/sec./cm.-5, p greater 0.05) were seen; these values returned to preoperative levels by the first postoperative day. After valve replacement, an elevation in the heart rate (HR) (preoperative 75 +/- 2 to 87 +/- 4 beats per minute, p less than 0.05) and a reduction in the stroke volume (SV) and stroke work index (SWI) (preoperative 35 +/- 2 to 26 +/- 2 Gm.-M. per square meter, p less than 0.05) occurred which persisted throughout the study. The endocardial viability ration (EVR) increased after valve replacement. These findings suggest that, following aortic valve replacement, (1) an increase HR is the mechanism utilized to maintain cardiac output, (2) subendocardial perfusion is enhanced as indicated by an increase in the EVR, and (3) the hemodynamic alterations following insertion of the porcine heterograft prosthesis are not different from those observed when other valves are placed in similar patient populations.