ABSTRACT. For genotyping of feline major histocompatibility complex (FLA) class II DRB, the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method using group-specific primers was tried. Sixty-six DRB genes were classified into 8 groups according to differences in the first 5' amino acid sequences. The group-specific primers were designed as forward ones, which were specific for 5' base sequences of genes in each group. Three to 7 appropriate restricted enzymes were selected by computer analysis for RFLP typing of the genes divided into each group. In 6 out of 9 cats, the results of DRB typed by direct sequence method agreed with results of the PCR-RFLP method using group-specific primers. In the other 3 cats, the number of genes amplified by group-specific primers was 1 or 2 more than those detected by direct sequence method. The direct sequence method in 9 cats identified 5 new FLA-DRB genes. The PCR-RFLP method using group-specific primers could divide 66 genes into 37 genes and 10 subgroups from the RFLP pattern. One to 6 genes in each cat, and a total of 203 genes and subgroups were detected in 68 domestic cats. The genes detected might be biased to the subgroup G1-1a (28.8%), DRB*0501 (10.3%), G1-2a (9.4%) and G6b (7.4%). The PCR-RFLP method using group-specific primers may be useful in typing FLA class II DRB. KEY WORDS: feline, FLA class II DRB genotyping, group-specific primer, PCR-RFLP method.J. Vet. Med. Sci. 62(12): 1283-1289, 2000 Major histocompatibility complex (MHC) class II molecules are heterodimeric glycoproteins involved in the regulation of the immune responses [21]. Human leukocyte antigen (HLA) class II genes consist of various components, DR, DP, DQ and etc. [11]. However, the structure of feline MHC (FLA) class II has not been well established, and only DR genes have been detected at the present time [29]. The DRB gene which encodes the β chain of DR molecule is the most polymorphic in class II genes in humans [13], pigs [8] and dogs [3]. Polymorphism of DRB gene accumulates at the second exon, which encodes the first extracellular domain, β 1 domain of the DR β chain [10]. The type of DRB gene is associated with the result of mixed lymphocyte reaction (MLR) [1], percent of 1-year renal graft survival [22], as well as susceptibility to autoimmune diseases [21,25] in humans. Polymerase chain reaction (PCR)-sequence specific oligonucleotide probe (SSOP) method or PCR-restriction fragment length polymorphism (RFLP) method has been used commonly to distinguish the genotypes of MHC class II [9,11]. It has also been reported that digestions of PCR products by some restriction endonucleases after the PCR for grouping of alleles by group-specific primers (modified PCR-RFLP method combined with group-specific primers) were used for genotyping of human DRB1 [16].Chronic renal failure (renal sclerosis) is one of the frequent and important diseases in feline medicine. The basic therapies for this failure are fluid therapy, and administrations of antibiotics, steroids,...

Transduction of the suicide gene of Herpes simplex virus thymidine kinase (Hsv-tk) into glioma cells or neural stem cells combined with pro-drug ganciclovir (GCV) treatment has been effective to treat experimental glioma in the rat through the bystander effect. Bone marrow stromal cells (MSCs) in the adult bone marrow have tropism for brain tumors and act as tumor stromal cells. Whether adult MSCs expressing Hsv-tk can also act as effector cells of the bystander killing effect on murine glioma cells was investigated. In vitro study of co-culture between 9L/LacZ (9L) glioma cells and Hsv-tk-transduced MSCs (MSCs/tk(＋)) followed by GCV administration in the culture medium resulted in apparent nuclear morphological changes in the 9L glioma cells surrounding the MSCs/tk(＋). 9L glioma cell survival in the presence of MSCs/tk(＋) and GCV treatment was quantitatively measured and showed significant decrease of 9L glioma cell proliferation with higher MSCs/tk(＋) ratio and GCV concentration. Intracerebral co-inoculation experiments in Fisher rats used 9L glioma cells and either MSCs/tk(＋) or Hsv-tk-non-transduced MSCs (MSCs/tk(-)) followed by intraperitoneal injection of GCV (100 mg/kg, daily for 7 days). The animals co-inoculated with 9L glioma cells and MSCs/tk(＋) showed significant retardation of tumor growth and prolongation of survival time compared with the animals with 9L glioma cells and MSCs/tk(-). Quantitative findings were established of the novel effects of adult MSCs/tk(＋) as effector cells of the bystander killing effect on glioma cells.

Magnesium has neuroprotective and antivasospastic properties in the presence of subarachnoid hemorrhage (SAH). The present study investigated the effect of intracisternal administration of magnesium on cerebral vasospasm in the experimental SAH rat model. The rat double-SAH model (0.2 mL autologous blood injected twice into the cisterna magna) was used. Normal saline (SAH group, N=8) or 10 mmol/L magnesium sulfate in normal saline (SAH + MG group, N=8) was infused into the cisterna magna at 1.5 microL/min for 30 min on day 5. Control rats without SAH also received intracisternal infusion of normal saline (control group, N=6). Local cerebral blood flow (CBF) at 24 locations and the weighted average were quantitatively measured by the autoradiographic technique using [(14)C]iodoantipyrine during infusion. The weighted average CBF was significantly reduced (P<0.01, Student's t-test) in the SAH group (0.78+/-0.16 mL g(-1) min(-1)) compared to the control group (1.0+/-0.15 mL g(-1) min(-1)) and was significantly improved (P<0.01, Student's t-test) in the SAH + MG group (0.98+/-0.18 mL g(-1) min(-1)). Local CBF was significantly reduced (P<0.05, unpaired t test) in 16 locations in the SAH group and significantly improved (P < 0.05, unpaired t test) in 12 locations in the SAH + MG group. Intracisternal infusion of magnesium sulfate significantly improved reduced CBF induced by experimental SAH in the rat.

Pseudohypoaldosteronism type 1 (PHA1) is a rare condition characterized by neonatal salt loss with elevated plasma aldosterone and renin levels. Two types of PHA1 have been described: an autosomal recessive systemic form and an autosomal dominant renal form, in which the target organ defect is confined to the renal tubules. The dominant renal form of PHA1 is caused by heterozygous mutations in the NR3C2 gene, which encodes the mineralocorticoid receptor (MR). We determined clinical and biochemical parameters in two familial and four sporadic Japanese patient and analyzed the status of the NR3C2 gene. Failure to thrive was noted in five of the six patients. In one of the familial cases, the mother had an episode of failure to thrive when she was a toddler, but received no medical treatment. NaCl supplementation was discontinued in four of the six patients after they reached one year of age and they have grown normally thereafter. However, in one patient, 9 g/day of salt has been required to maintain serum Na concentration after 1 year of age. Analysis of NR3C2 identified three novel mutations [c. C1951T (p.R651X), c.304_305delGC (p.A102fsX103), c.del 603A (p.T201fsX34)] and one previously reported mutation [c.A2839G (p.947X)]. p.R651X was identified in one familial case and one unrelated sporadic patient. The patient who has been supplemented with large amount of salt was heterozygous for c.del 603A in exon 2. In conclusion, our study expands the spectrum of phenotypes, and characterized mutations of NR3C2 in the renal form of PHA1.

The effect of transplantation of adult bone marrow stromal cells (MSCs) into the freeze-lesioned left barrel field cortex in the rat was investigated by measurement of local cerebral glucose utilization (lCMR(glc)) in the anatomic structures of the whisker-to-barrel cortex sensory pathway. Bone marrow stromal cells or phosphate-buffered saline (PBS) were injected intracerebrally into the boundary zone 1 h after induction of the freezing cortical lesion. Three weeks after surgery, the 2-[(14)C]deoxyglucose method was used to measure lCMR(glc) during right whisker stimulation. The volume of the primary necrotic freezing lesion was significantly reduced (P<0.05), and secondary retrograde degeneration in the left ventral posteromedial (VPM) thalamic nucleus was diminished in the MSC-treated group. Local cerebral glucose utilization measurements showed that the freezing cortical lesion did not alter the metabolic responses to stimulation in the brain stem trigeminal nuclei, but eliminated the responses in the left VPM nucleus and periphery of the barrel cortex in the PBS-treated group. The left/right (stimulated/unstimulated) lCMR(glc) ratios were significantly improved in both the VPM nucleus and periphery of the barrel cortex in the MSC-treated group compared with the PBS-treated group (P<0.05). These results indicate that MSC transplantation in adults may stimulate metabolic and functional recovery in injured neuronal pathways.