DLA-DRB1 polymorphisms in dogs defined by sequence-specific oligonucleotide probes (SSOP)

To date, DNA sequences for 29 dog DLA-DRB1 alleles have been reported. However, no data exists on the frequencies of these alleles within the general dog population, nor is there any indication of whether there is interbreed variation of allele distribution. We have addressed this by establishing a molecular based sequence-specific oligonucleotide probing (SSOP) method to identify all of the known broad DRB1 types and we have used this to type a random panel of dogs. A series of oligonucleotide probes were designed to detect known polymorphisms in the three DRB1 hypervariable regions, together with two distinctive motifs in other regions of exon 2. This set of probes enabled us to assign broad DRB1 types. Two hundred and eighteen dogs were SSOP typed for DRB1. All but 4 of the published DLA-DRB1 alleles were identified in these animals. Interbreed variation in both allele distributions and allele frequencies were observed, which may be useful in the study of genetic variation between breeds. This variation also has implications for the selection of control groups for studies aimed at identifying MHC associations with disease susceptibility in the dog.     

Direct ex vivo analysis reveals distinct phenotypic patterns of HIV-specific CD8(+) T lymphocyte activation in response to therapeutic manipulation of virus load

Therapeutic intervention with antiretroviral therapy (ART) enables the modulation of HIV virus load and hence provides a unique opportunity to study the consequences of varying antigen load on the phenotype of virus-specific CD8(+) T lymphocytes in a persistent human viral infection. The recent advent of tetrameric peptide / HLA class I complexes has enabled the direct phenotypic characterization of antigen-specific T cell populations ex vivo. Here, we use this technology to examine directly ex vivo the consequences of therapeutic manipulation of HIV virus load on the phenotype of HIV-specific CTL. Our observations show that: (1) distinct sequential activation patterns of CD8(+) T cells are associated with increasing virus load; (2) T cell receptor (TCR) down-regulation without apoptosis represents an early event during the generation of a T cell response in a natural infection and precedes the emergence of two distinct antigen-specific CD8(+) T cell populations which differ in TCR and CD8 expression levels. Clear differences in surface Annexin V staining were observed between these populations. The observation that CTL activation, demonstrated by TCR and CD8 down-regulation, in response to rising levels of virus load, co-segregates with apoptosis only during later stages of the response indicates that antigen-associated cell death is restricted to distinct subpopulations of CTL.     

Substance P and somatostatin metabolism in sympathetic and special sensory ganglia in vitro

Mechanisms regulating the content of the putative peptide transmitters, substance P and somatostatin, were examined in several neuronal populations in culture. Substance P levels increased more than 25-fold within 48 h in sympathetic neurons in the explanted rat superior cervical ganglion, and remained elevated for 4 weeks. Identity of the peptide was authenticated by combined high pressure liquid chromatography-radioimmunoassay. Veratridine prevented the increase of substance P in vitro, and tetrodotoxin blocked the veratridine effect, suggesting that sodium ion influx and membrane depolarization prevent peptide elevation. Veratridine (or potassium)-induced membrane depolarization released substance P into the culture medium through a calcium-dependent process. Consequently, at least some veratridine effects are attributable to release and subsequent depletion of ganglion peptide. However, the inhibitory effects of veratridine were far greater than could be accounted for by the quantity of peptide released, suggesting a separate influence on net synthesis (synthesis less catabolism) of substance P. Viewed in conjunction with previous in vivo studies, our observations suggest that trans-synaptic impulses, through the mediation of postsynaptic sodium flux, release substance P from sympathetic neurons and also regulate intracellular peptide metabolism. To determine whether the processes regulating substance P in sympathetic neurons reflect generalized mechanisms, a different peptide, somatostatin, was examined in sympathetic neurons; moreover, substance P was examined in a different neuronal population, special sensory neurons in the nodose ganglion. Substance P levels increased significantly in both sympathetic and sensory neurons after explantation, and somatostatin levels increased in sympathetic neurons. In each instance, the increase was dependent upon the presence of the calcium ions. Moreover, these increases were all prevented by veratridine, in a tetrodotoxin-sensitive manner. Our observations suggest that common regulatory mechanisms govern peptide transmitter metabolism in diverse neuronal populations.     

Effects of affirmative action in medical schools. A study of the class of 1975

In the early 1970s, affirmative-action programs were introduced to accomplish a number of social goals, including increasing the supply of minority physicians and improving the health care of the poor. To assess the success of such programs, we analyzed data on people who graduated from U.S. medical schools in 1975 to determine how specialty choice, practice locations, patient populations served, and board-certification rates differ between minority and nonminority graduates. A larger proportion of minority graduates (55 per cent vs. 41 per cent, P less than 0.001) chose the primary-care specialties of family practice, general internal medicine, general pediatrics, and obstetrics-gynecology. Significantly more minority physicians (12 per cent vs. 6 per cent, P less than 0.01) practiced in locations designated as health-manpower shortage areas by the federal government and had more Medicaid recipients in their patient populations (31 per cent for blacks, 24 per cent for Hispanics, 14 per cent for whites; P less than 0.001). Physicians from each racial or ethnic group served disproportionately more patients of their own racial or ethnic group (P less than 0.001), but minority physicians did not serve significantly more persons from other racial or ethnic minority groups than did nonminority physicians. Many minority physicians served patient populations much like those of their nonminority colleagues, which indicates that substantial integration of the medical marketplace has taken place. Significantly fewer minority graduates had become board-certified by 1984 (48 per cent vs. 80 per cent, P less than 0.001), and most of this disparity was associated with differences in premedical-school characteristics and in the patient populations they served. Our analysis shows that minority graduates of the medical school class of 1975 are fulfilling many of the objectives of affirmative-action programs.     

B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma involving bone marrow with an interfollicular pattern

B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) may involve the bone marrow in nodular, interstitial, diffuse, or mixed patterns. However, B-cell CLL/SLL associated with large reactive germinal centers (the so-called interfollicular pattern) involving the bone marrow is not reported. We describe 2 examples of B-cell CLL/SLL that subtotally replaced the bone marrow with an interfollicular pattern. In both cases, the neoplasms were composed of small round lymphoid cells; proliferation centers also were present. The neoplasms surrounded large reactive germinal centers that were devoid of peripheral mantle zones. The germinal centers were paratrabecular and nonparatrabecular in case 1 and nonparatrabecular in case 2. Flow cytometry immunophenotypic studies done on bone marrow aspiration samples of both cases showed a uniform population of neoplastic cells positive for pan-B-cell antigens and the CD5 and CD23 antigens. Immunohistochemical studies done on bone marrow biopsy sections supported the flow cytometry results and demonstrated that the germinal centers were negative for BCL-2. B-cell CLL/SLL may rarely involve the bone marrow with an interfollicular pattern. Knowledge of this pattern will prevent confusion with follicle center lymphoma and large cell transformation, both of which initially were considered in the differential diagnosis of these cases.     

Binding proteins for insulin-like growth factors in the human ovary: identification, follicular fluid levels and immunohistological localization of the 29-32 kd type 1 binding protein, IGF-bp1.

The occurrence of pregnancy-associated endometrial alpha 1-globulin (alpha 1-PEG), a 29-32 kd insulin-like growth factor binding protein, now termed type 1 or IGF-bp1, has been examined in the human ovary by monoclonal and polyclonal antibody based radioimmunoassay and immunohistological techniques. Follicular fluids aspirated from 51 follicles of 32 women undergoing hyperstimulation involving buserelin or clomiphene-based protocols contained 35.5-276.0 ng/ml (mean 101.0 mg/ml) of immunoreactive IGF-bp1. Mean fluid concentrations were three times the level of IGF-bp1 detected in paired serum samples, available for 21 women. Immunoreactive IGF-bp1 in follicular fluid exhibited similar dose-response curves to purified protein and amniotic fluid and immunoreactive IGF-bp1 coeluted in gel filtration with a peak of [125I]-IGF-1 binding corresponding to the elution profile of purified IGF-bp1. Gel filtration also revealed the presence in follicular fluid of a greater than 100 kd binding protein with a binding capacity equal to IGF-bp1 under the conditions employed. A highly significant correlation (P less than 0.001) was found between follicular fluid progesterone and IGF-bp1 and a correlation of lower significance was found between oestradiol and IGF-bp1 (P less than 0.05). However, only low levels of immunoreactive IGF-bp1 were detected in supernatant media of granulosa cells in culture (range undetectable to 2.3 ng/ml). Employing monoclonal antibody-based immunohistology, immunoreactive IGF-bp1 was consistently associated with luteinized granulosa cells of corpora lutea rather than paraluteal cells and its intensity of reactivity appeared to reflect luteal phase steroid hormone profiles. No consistent reactivity was detected in preovulatory follicles and granulosa cells in culture, although reactivity was associated with primordial oocytes. Immunoreactive IGF-bp1 was detected in six of nine supernatant media of explants of luteal tissue obtained from five corpora lutea, with levels ranging from undetectable to greater than 200 ng/ml. These observations suggest that IGF-bp1 is primarily related to luteinization of the granulosa and the resultant luteal cells, and if produced by the luteal cells, additional exogenous factors are required to induce production by granulosa cells in vitro.     

Gel scintillation counting of I131 in plasma at high efficiencies

A method is described for assaying low concentrations of (131)I in plasma by detecting the beta radiation in a gel scintillator. Higher detection efficiencies are obtained compared with measurements of the gamma radiation from this radioisotope using a well-type crystal of sodium iodide.     

Contraindications to lumbar puncture as defined by computed cranial tomography.

Papilloedema is not always an adequate predictor of potential complications from lumbar puncture, and many clinicians are using computed tomography (CT) before lumbar puncture in an effort to identify more accurately the "at risk" patient. This paper identifies the following anatomical criteria defined by CT scanning that correlate with unequal pressures between intracranial compartments and predispose a patient to herniation following decompression of the spinal compartment: lateral shift of midline structures, loss of the suprachiasmatic and basilar cisterns, obliteration of the fourth ventricle, or obliteration of the superior cerebellar and quadrigeminal plate cisterns with sparing of the ambient cisterns. These criteria should be considered to be contraindications to lumbar puncture.