The second generation of the International Equine Gene Mapping Workshop half-sibling linkage map.

A low-density, male-based linkage map was constructed as one of the objectives of the International Equine Gene Mapping Workshop. Here we report the second generation map based on testing 503 half-sibling offspring from 13 sire families for 344 informative markers using the CRIMAP program. The multipoint linkage analysis localized 310 markers (90%) with 257 markers being linearly ordered. The map included 34 linkage groups representing all 31 autosomes and spanning 2262 cM with an average interval between loci of 10.1 cM. This map is a milestone in that it is the first map with linkage groups assigned to each of the 31 automosomes and a single linkage group to all but three chromosomes.

A phase I trial of AUC-directed carboplatin with infusional doxorubicin and ifosfamide plus G-CSF in patients with advanced gynecologic malignancies.

The effect of the addition of G-CSF to carboplatin, ifosfamide and doxorubicin (CIA) at the maximally tolerated dose (MTD) was studied in a phase I clinical trial. Nine patients with incurable solid tumors were treated: six endometrial and epithelial ovarian cancers, one colon cancer with pelvic masses and two unknown primary cancers. The carboplatin dose was calculated using the Calvert formula and administered in a standard 30-min intravenous infusion. The initial carboplatin dose was AUC 4.0 mg/ml per min. Fixed doses of ifosfamide (1.25 g/m2 per day), mesna (1.0 g/m2 per day, and doxorubicin (15 mg/m2 per day) were combined and given as a 4-day continuous intravenous infusion in an attempt to decrease nonhematologic toxicity. The dose-limiting toxicity of CIA was myelosuppression, mainly neutropenia and thrombocytopenia. Nonhematologic toxicities were hemorrhagic cystitis, weakness, fatigue, and nausea and vomiting. The MTD for CIA was established at the first dose level of carboplatin (4.0 mg/ml per min). Following this, G-CSF was added to the regimen in an unsuccessful effort to escalate the carboplatin dose. Free and total carboplatin pharmacokinetics were determined using flameless atomic absorption spectroscopy. There was one complete response and one partial response among eight evaluable patients. Both responding patients had advanced ovarian cancer. We conclude that carboplatin dose intensification beyond an AUC of 4.0 mg/ml per min is not made feasible by the addition of G-CSF to infusional doxorubicin and ifosfamide in patients with advanced gynecologic cancer.

Report of the International Equine Gene Mapping Workshop: male linkage map.

The goal of the First International Equine Gene Mapping Workshop, held in 1995, was the construction of a low density, male linkage map for the horse. For this purpose, the International Horse Reference Family Panel (IHRFP) was established, consisting of 12 paternal half-sib families with 448 half-sib offspring provided by 10 laboratories. Blood samples were collected and DNA extracted in each laboratory and sent to the Lexington laboratory (KY, USA) for dispatch in aliquots to 14 typing laboratories. In total, 161 markers (144 microsatellites, seven blood groups and 10 proteins) were tested for all families for which the sire was heterozygous. Genealogies and typing data were sent for analysis to the INRA laboratory (Jouy-en-Josas, France) according to a specific format and entered into a database with input verification and output processes. Linkage analysis was performed with the CRIMAP program. Significant linkage was detected for 124 loci, of which 95 were unambiguously ordered using a multipoint analysis with an average spacing of 14.2 CM. These loci were distributed among 29 linkage groups. A more comprehensive analysis including synteny group data and FISH data suggested that 26 autosomes out of 31 are covered. The complete map spans 936 CM.

IL-12, as an adjuvant, promotes a T helper 1 cell, but does not suppress a T helper 2 cell recall response.

IL-12 is a potent inducer of NK and cytolytic T cell activity, IFN-gamma production, and T cell proliferation, and is necessary for differentiation of naive T cells to the Th1 subset. We have previously shown that IL-12 promotes a primary Th1 response and suppresses a primary Th2 response in lymph nodes of mice primed with a model hapten-protein conjugate, 2,4,6-trinitrophenyl (TNP)-keyhole limpet hemocyanin (KLH). We have now extended these studies to determine the Th phenotype of the recall response following immunization with soluble Ag and IL-12. For these experiments, mice were primed with TNP-KLH with or without treatment with IL-12, allowed to progress beyond the primary immune response, and challenged by i.p. injection of TNP-KLH. The phenotype of the recall response was monitored by measuring ex vivo production of IFN-gamma and IL-4 in Ag-stimulated lymph node and spleen cell cultures. Titer and isotype of TNP-specific serum Abs were also evaluated. Mice primed with Ag+IL-12 developed a Th1 recall response, as detected by KLH-specific IFN-gamma production from cultured spleen cells and the presence of TNP-specific IgG2a Ab in serum. However, they also developed an Ag-specific Th2 recall response, as characterized by Ag-induced IL-4 production from spleen cells and the presence of high titers of anti-TNP IgG1 in the serum. Studies of the cytokine profile during the primary response revealed that IL-12 induced in spleen cells the capacity to express both IL-4 and IFN-gamma. CD4+ T cells are necessary for production of IL-4 in the spleens of IL-12-treated mice, and most likely account for the Th2 recall response detected in mice primed with Ag+IL-12. These results indicate that the Th1 phenotype induced by immunization with IL-12 and Ag is maintained so that a Th1 recall response is expressed upon subsequent challenge with Ag. However, immunization with IL-12 also supports the development of a Th2 recall response, indicating that the Th1-inducing effect of IL-12 in vivo is not accompanied by a long lasting suppression of Th2 development.

Immunoglobulin isotype modulation after administration of IL-12.

We have begun a series of experiments assessing the role of IL-12 in the humoral immune response. IL-12 is known to enhance cellular immunity causing a shift toward a Th1, as opposed to a Th2, response. IL-12 is also a potent stimulator of IFN-gamma production which, among other activities, modulates isotype expression particularly with respect to IgG2a. We have performed a series of experiments involving the concurrent dosing of mice with murine IL-12 and TNP-KLH followed by the monitoring of IgG1 and IgG2a anti-TNP responses and total IgG1 and IgG2a levels. Following administration of IL-12, specific anti-TNP titers showed an IgG2a increase while IgG1 responses were markedly lower than those exhibited by animals which did not receive IL-12. Total IgG1 levels in IL-12 treated mice remained at or near baseline while untreated mice demonstrated an increase in total IgG1 levels. In addition, lymph nodes from these mice were removed, stimulated with KLH and assayed for expression of murine IFN-gamma and IL-4. Murine IFN-gamma levels in supernatants obtained from IL-12 treated mice were elevated over those seen in untreated mice while IL-4 levels were suppressed.

Prenatal sex determination by fluorescent staining of the cervical smear for the presence of a Y chromosome: an evaluation.

PIP: If the parents do not want to take the 50% chance that a male fetus will be involved with an x-linked disease, it is desirable to predict fetal sex early enough to safely terminate pregnancy. Slides obtained from midcervical smears of 20 consecutive patients at different stages of gestation were stained with .5% aqueous solution of quinacrine hydrochloride then dipped 3 times in a buffer with pH of 5.5 and mounted wet. On each slide 100 cells were studied with fluorescent light. In none was the characteristic fluorescent y-body seen. As there was no follow-up, an additional 18 patients were similarly studied. 7 delivered male infants in spite of the absence of a y-body on cervical smears. It is concluded that this technique is not satisfactory for prenatal sex determination.^ieng