Establishment and characterization of an in vitro model of acquired resistance to cisplatin in a human testicular nonseminomatous germ cell line.

Clinically, human testicular nonseminomatous germ cell tumors exhibit remarkable sensitivity to platinum-based chemotherapy. To define better the mechanistic basis for this unusual sensitivity, the biochemical determinants of platinum-induced cytotoxicity have been investigated in a human testicular tumor cell line (GCT27) established from a previously untreated patient and in an in vitro derived 5.6-fold cisplatin-resistant stable variant (GCT27cisR). Compared to 12 ovarian and 5 cervical human tumor cell lines, the parent GCT27 line was among the most sensitive to the cytotoxic effects of both cisplatin (dosage producing 50% inhibition, 0.2 microM) and carboplatin (dosage producing 50% inhibition, 2.9 microM), thus reflecting clinical data. A 4-day exposure sulforhodamine B-staining assay was used to determine that GCT27cisR was cross-resistant to carboplatin and iproplatin and the classical bifunctional alkylating agents melphalan and chlorambucil. Partial cross-resistance was observed to tetraplatin, methotrexate, and mitomycin C. No cross-resistance was observed to Adriamycin, etoposide, vinblastine, bleomycin, 1-beta-D-arabinofuranosylcytosine, and 5-fluorouracil. Intracellular cisplatin accumulation across the dose range 2.5-100 microM (for 2 h) was 1.6 +/- 0.39-fold (mean +/- SD) greater for the parent line. There was no significant difference in glutathione levels between the two lines. The acquired resistance line was 1.9-fold more resistant than the parent line to the cytotoxic effects of cadmium chloride. There was no significant difference between the two lines, however, in the total amounts of platinum bound to DNA after cisplatin exposure (25, 50, or 100 microM for 2 h). The removal of total platinum adducts from DNA was significantly faster for GCT27cisR compared to the parent line (half-times of removal, 32 and 67 h, respectively). These data suggest that the abnormal sensitivity of the parent testicular tumor cell line to platinum-containing anticancer drugs may be due predominantly to an inherent defect in the ability of these cells to remove platinum from their DNA. This defect is apparently lost in the acquired resistance counterpart. Reduced intracellular accumulation and increased cytoplasmic concentrations of metallothionein may also contribute, in part, to the acquisition of cisplatin resistance in this model.     

A quantitative analysis of valgus torque on the ACL: a human cadaveric study.

The loads needed to elicit a positive pivot shift test in a knee with an anterior cruciate ligament (ACL) rupture have not been quantified. The coupled anterior tibial translation (ATT), coupled internal tibial rotation (ITR), and the in situ force in the ACL in response to a valgus torque, an inherent component of the pivot shift test, were measured in 10 human cadaveric knee specimens. Using a robotic/universal force-moment sensor testing system, valgus torques ranging from 0.0 to 10.0 Nm were applied in nine increments on the intact and ACL-deficient knee in flexion ranging from 0 degrees to 90 degrees. At 15 degrees of knee flexion, the coupled ATT and ITR were significantly increased in the ACL-deficient knee when compared to the intact knee. Coupled ATT increased a maximum of 291% (6.7 mm, p<0.05), while coupled ITR increased a maximum of 85% (5.1 degrees, p<0.05). At 30 degrees, the increases in coupled ATT and ITR were significant at valgus loads of 3.3 Nm and greater with a maximum increase in coupled ATT of 137% (6.3 mm, p<0.05) and a maximum increase in coupled ITR of 38% (3.6 degrees, p<0.05). At 45 degrees, coupled ATT increased significantly (maximum of 69%, 4.4 mm, p<0.05), but only at torques > or =6.7 Nm. The in situ force in the ACL was less than 20 N for all flexion angles when a torque between 3.3 and 5.0 Nm was applied. Low valgus torque elicited tibial subluxation in the ACL-deficient knee with low in situ ACL forces, similar to a positive pivot shift test. Thus, application of a valgus torque may be suitable to evaluate ACL-deficient and ACL-reconstructed knees, since subluxation can be achieved with minimal harm to the ACL graft. This work is important in understanding one load component needed for the pivot shift examination; further studies quantifying other load components are essential for better comprehension of the in vivo pivot shift examination.     

Reduced levels of IgG subclasses and IgA in young children with asthma

Serum immunoglobulins including IgG subclasses were measured in 73 unselected children with asthma. The results showed that 22 (30%) had partial IgA and/or IgG₄ subclass deficiency. Clinical assessment showed that 21 children were infection-prone, and 52 were not. Further analysis showed that infection-prone children were significantly different from non-infection-prone children with regard to familial history of allergy (29% vs 60%, p = 0.015), elevated IgE (62% vs 33%, p = 0.021), IgA deficiency (38% vs 15%, p = 0.38) and IgG subclass deficiency (24% vs 4%, p = 0.018). These results suggest that there may be subgroups of children with asthma who are also immunodeficient.     

INVOLVEMENT OF NON-NMDA AND NMDA RECEPTORS IN GLUTAMATE-INDUCED PRESSOR OR DEPRESSOR RESPONSES OF THE PONS AND MEDULLA

1. Fifty-five intact and six baroreceptor denervated and vagotomized cats of either sex were anaesthetized intraperito-neally with urethane (400 mg/kg) and a-chloralose (40 mg/kg). Responses of the systemic arterial pressure (SAP), mean SAP (MSAP) and sympathetic vertebral nerve (VNA) and renal nerve activities (RNA) were recorded. 2. In intact animals, monosodium L-glutamate (Glu, 0.1 mol/L, 50 nL) was microinjected into pressor areas of the locus coeruleus (LC), gigantocellular tegmental field (GTF), rostral ventrolateral medulla (RVLM) and dorsomedial medulla (DM), and the depressor areas of caudal ventrolateral medulla (CVLM). The induced actions were compared before and after microinjection of either glutamate antagonists, glutamate diethylester (GDEE, 0.5 mol/L, 50–100nL), a competitive AMPA receptor blocker, or 2-amino-5-phosphonovaleric acid (D-AP5, 0.025 mol/L, 50–100 nL), a competitive N-methyl-D-aspartate (NMDA) receptor blocker. GDEE completely blocked the increases of SAP and VNA elicited from all pressor areas. D-AP5 only partially blocked the pressor but slightly blocked VNA and RNA responses from LC, GTF and DM, particularly those from RVLM. Neither GDEE nor D-AP5 blocked the depressor responses of SAP and two nerve activities elicited from CVLM. 3. In baroreceptor denervated animals, NMDA (2 mmol/L, 50–100 nL) and AMPA (0.2 mmol/L, 50–100 nL) were micro-injected into the same pressor areas of GTF, RVLM and DM and the depressor area of CVLM responsive to Glu activation (0.1 mol/L, 30 nL). In RVLM, DM and CVLM, the results of either NMDA or AMPA were similar to those induced by Glu. However, in GTF, microinjection of either NMDA or AMPA did not induce similar responses to Glu. This suggests that the nature of GTF may differ from RVLM and DM. 4. The above results suggest that the Glu-induced pressor responses from LC, GTF, DM and especially RVLM, are primarily mediated through AMPA receptors. The Glu-induced depressor responses from CVLM may not be predominantly mediated by either AMPA or NMDA receptors. 5. In both baroreceptor-intact and -denervated cats stimulation of the pressor areas often produced an increase of VNA and a decrease of RNA, while in the depressor CVLM decreased both VNA and RNA. The VNA, but not RNA were positively correlated with the pressor responses, while both VNA and RNA were positively correlated with the depressor responses. This may suggest that neurons of the sympathetic vertebral and renal nerves are topographically organized in the brain.