Effect of vitamin D supplementation on pegylated interferon/ribavirin therapy for chronic hepatitis C genotype 1b: a randomized controlled trial.

Chronic HCV-infected patients tend to have vitamin D deficiency, suggesting that vitamin D supplementation may enhance the efficacy of treatment with pegylated interferon (PEG-IFN) and ribavirin (RBV). We therefore assessed the effects of vitamin D supplementation on viral response to PEG-IFN/RBV. Eighty-four patients with HCV genotype 1b were randomized, 42 to oral vitamin D supplementation (1000 IU/day) and 42 to nonsupplementation (control), from week 8 to the end of PEG-IFN/RBV therapy. The primary end point was undetectable HCV RNA at week 24 (viral response [VR]). VR rate at week 24 was significantly higher in the vitamin D than in the control group (78.6% vs 54.8% P = 0.037). Adverse events were similar in both groups. When patients were subdivided by IL28B SNP rs8099917 genotype, those with the TT genotype group showed a significantly higher VR rate at week 24 with than without vitamin D supplementation (86.2% vs 63.3% vs P = 0.044). Although patients with the TG/GG genotype, who were relatively resistant to PEG-IFN treatment, had similar VR rates at week 24 with and without vitamin D supplementation, the decline in viral load from week 8 to week 24 was significantly greater with than without vitamin D supplementation. Multivariate analysis showed that rs8099917 genotype and vitamin D supplementation contributed significantly to VR at week 24. SVR rates were similar in the vitamin D and control groups [64.3% (27/42) vs 50% (21/42), P = 0.19]. Vitamin D supplementation may enhance the effects of PEG-IFN/RBV in HCV genotype 1b-infected patients.

Characterization of defectiveness in endogenous antigen presentation of novel murine cells established from methylcholanthrene-induced fibrosarcomas.

Three cell lines (4A1, 4C2 and 6D1 cells) derived from fibrosarcoma induced by the inoculation of 3-methylcholanthrene into C3H/HeN (H-2k) mice were examined for their ability to present antigens to CD8+ cytotoxic T lymphocytes (CTL). 6D1 and 4C2 cells were deficient in presenting endogenously synthesized influenza virus antigens to CTL, but they were able to present antigens when they were sensitized with a synthetic epitope peptide. The expression of the H-2 Kk gene in 4C2 and 6D1 cells was much reduced and was detectable only with Northern blot hybridization. The expression of two transporter genes (TAP1 and TAP2), examined by Northern hybridization, was also reduced in both cells, and negligible particularly in 4C2 cells. Interferon-gamma (IFN-gamma) treatment of these cells induced expression of Kk, TAP1 and TAP2 genes and rescued the defect of class I-restricted antigen presentation in 4C2 and 6D1 cells. Even after this treatment, however, antigen-presentation capability of 4C2 cells was still much lower than that of normal 4A1 cells. This finding suggests that 4C2 cells might have an additional defective gene(s), whose products are involved in the processing of class I-restricted antigen, besides the Kk and TAP genes, and this may explain the difficulty of 4C2 cells to induce tumour-specific immunity, as described previously. To our knowledge, the 4C2 cell is the first tumour cell postulated to have more than three defective genes involved in class I-restricted antigen presentation.

[Statistical analysis of relationship between microalbuminuria and diabetic retinopathy].

The relationship between microalbuminuria indicated by the logarithm of the albumin index and the stage of diabetic retinopathy was investigated using 175 diabetic subjects. The relationship and its dependence on the duration and the age of onset of diabetes were analyzed statistically with logistic regression. In younger-onset subjects, microalbuminuria was strongly related to the stage of retinopathy, but in older-onset subjects, the relationship showed to lack. For each subject, the frequency of retinopathy was predicted by the estimated probability calculated with the regression model. When the critical probability was 50%, the sensitivity and specificity were 53.1% and 76.2%, respectively. These results indicated that the regression model using the albumin index might be a useful method to predict the frequency of diabetic retinopathy even without ophthalmoscopic examination.

[CT analysis of pulmonary emphysematous and fibrotic changes in patients with von Recklinghausen's disease].

Five cases of von Recklinghausen's disease were studied with regard to their association with chest diseases, especially pulmonary emphysematous and fibrotic changes. CT analysis revealed that four patients out of five had multiple pulmonary emphysematous lesions and that those lesions were seen in all segments. In two cases out of the four, it was difficult to detect their abnormalities by an ordinary chest roentgenogram, suggesting the possibility of higher incidence of emphysematous changes in von Recklinghausen's disease than has been expected. On the other hand, pulmonary fibrosis was observed in two cases. There were two patients who had no fibrosis but emphysematous lesions. TBLB was performed in one case, in whom no fibrotic changes were observed. Usually pulmonary fibrosis is associated with emphysematous bullae in patients with von Recklinghausen's disease. However, fibrotic and emphysematous changes may not always occur in combination in von Recklinghausen's disease.

Enhanced immunogenicity of the cultured rat fibrosarcoma KMT-17 by cultivation in a low concentration of fetal calf serum.

We examined in different culture conditions alterations in the tumorigenicity and immunogenicity of an A3 clone that had been derived from a rat fibrosarcoma KMT-17. When a fetal calf serum concentration in a culture medium was lowered from 10 to 1%, the tumorigenicity was diminished while the immunogenicity was enhanced in a reversible manner; this was accompanied by a reversible prolongation of the in vitro doubling time. These phenomena were not due to an increase in the quantities of the original tumor-associated antigen and/or of the rat major histocompatibility complex (RT1) but seemed to be due to the appearance of a unique antigen(s) that was detected by an antibody taken from rats immunized with A3 tumor cells cultured in the low fetal calf serum concentration; this antigen(s) may consist of glycoprotein and exist as a crypt antigen(s). These phenomena were measured by an absorption test and flow cytometric analysis. Our observations suggest that the in vitro culture condition of tumor cells, in particular their culturing in the low fetal calf serum concentration medium, modifies the surface of tumor cells and causes a diminishment in their tumorigenicity and an enhancement of their immunogenicity.

[Macrophage-mediated induction of drug-resistance variants in a mouse mammary tumor cell line].

The ability of macrophages to induce drug-resistant mutants was studied in an in vitro, macrophage-tumor cell coculture system utilizing the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus as measured by resistance to 6-thioguanine. Tumor cells of the metastatic mouse mammary tumor line 66 were sensitive to macrophage induction of thioguanine resistance as shown by an increase in the frequency of thioguanine resistant variants which arose following macrophage coculture to levels at least 5 to 10 fold above the spontaneous frequency. This increased frequency was not seen in a series of related, generally nonmetastatic lines. Detection of increased numbers of variants depended upon the macrophage to tumor cell ratio, with 50:1 or greater being necessary. The activity of the macrophages was dependent upon their activation stage. The induction of drug-resistant variants could be inhibited by oxygen radical scavengers. The basis for the emergence of thioguanine resistant cells appeared to be induction of new variants rather than selection of pre-existing resistant cells from the parental population since thioguanine sensitive and resistant cells were equally sensitive to macrophage mediated toxicity. In 6 of the 6 macrophage-induced variants tested, resistance was associated with loss of HGPRT activity. The reverse mutation frequency rate at the HGPRT locus in 5 macrophage-induced variants was low and similar to that of a stable, ethyl methanesulfonate-induced thioguanine resistant line, suggesting that macrophage induction of thioguanine resistance was the result of a true mutation, rather than an epigenetic event. Macrophages isolated directly from growing mammary tumors, as well as activated peritoneal macrophages, were capable of inducing thioguanine resistance in line 66 cells.

Macrophage-mediated induction of drug-resistant variants in a mouse mammary tumor cell line.

The ability of macrophages to induce drug-resistant variants was studied in an in vitro macrophage-tumor cell coculture system utilizing the hypoxanthine-guanine phosphoribosyl transferase locus as measured by resistance to 6-thioguanine. Tumor cells of mouse mammary tumor line 66 were sensitive to macrophage induction of thioguanine resistance as shown by an increase in the frequency of thioguanine-resistant variants which arose following macrophage coculture to levels at least 5- to 10-fold above the spontaneous frequency. Detection of increased numbers of variants depended upon the macrophage:tumor cell ratio, with 50:1 or greater being necessary. The activity of the macrophages was dependent upon their activation stage. The induction of drug-resistant variants could be inhibited by oxygen radical scavengers. The basis for the emergence of thioguanine-resistant cells appeared to be induction of new variants rather than selection of preexisting resistant cells from the parental population, since thioguanine-sensitive and -resistant cells were equally sensitive to macrophage-mediated toxicity. In six of the six macrophage-induced variants tested, resistance was associated with loss of hypoxanthine-guanine phosphoribosyl transferase activity. The reverse variation frequency at the hypoxanthine-guanine phosphoribosyl transferase locus in five macrophage-induced variants was low and similar to that of a stable ethyl methanesulfonate-induced, thioguanine-resistant line. Macrophages isolated directly from growing mammary tumors, as well as activated peritoneal macrophages, were capable of inducing thioguanine resistance in line 66 cells.

Development of highly immunogenic variants of a rat fibrosarcoma line during in vitro cultivation.

Rat fibrosarcoma KMT-17 cells decreased in tumorigenicity when cultured in vitro. Eight clones derived from cultured KMT-17 cell lines (c-KMT-17) were examined for their tumorigenicity, immunosensitivity, and immunogenicity. All the clones were less or nontumorigenic in normal syngeneic rats than KMT-17 cells maintained in vivo. All eight clones produced tumors in rats immunosuppressed with 600 rad 60Co; differences in degree of tumorigenicity were seen among clones in rats irradiated with 250 rad 60Co. Although immunosensitivity of the eight clones to complement-dependent and cell-mediated cytotoxicity was the same or less than that of KMT-17 cells, al leight clones induced greater transplantation resistance to KMT-17 than KMT-17 itself. Cold target inhibition tests demonstrated new antigens in a highly immunogenic variant in addition to the original tumor associated antigen (TAA). New glycolipids, not observed in KMT-17 cells, were demonstrated in the clones by thin layer chromatography. These results suggest that new antigens appearing during culture of KMT-17 may act as helper antigens for TAA, increasing the immunogenicity and decreasing the tumorigenicity of the cultured cells.

Differential sensitivity of metastatic versus nonmetastatic mammary tumor cells to macrophage-mediated cytostasis.

Sensitivity to macrophage-mediated cytostasis was determined with 4 tumor cell lines derived from a single, spontaneously arising mouse mammary tumor. Cytostasis was measured in a 48-hour [3H]thymidine-incorporation assay with the use of maleic vinyl ether (pyran) fraction 2 (MVE-2)-elicited peritoneal macrophages as effector cells. Metastatic tumor lines 66 and 410.4 were less sensitive than nonmetastatic lines 67 and 168. Pretreatment of tumor cells with indomethacin for 24 hours before assay increased the cytostatic sensitivity of the metastatic tumor lines but did not affect that of the nonmetastatic tumor lines. Addition of 100 ng lipopolysaccharide (LPS)/ml to the assay mixture of MVE-2-primed macrophages and tumor cells or pretreatment of macrophages with LPS markedly lessened the differences in cytostatic sensitivity among the metastatic and nonmetastatic lines. Pretreatment of tumor cells with indomethacin plus addition of LPS during the effector phase of the assay completely abrogated differences in sensitivity. These results suggest that differences in sensitivity of metastatic versus nonmetastatic tumor cells to macrophage cytostasis are due to both tumor cell (prostaglandin) and effector cell (activation state) factors.

Correlation of frequency of induced mutation and metastatic potential in tumor cell lines from a single mouse mammary tumor.

Spontaneous mutation rates were determined in mouse mammary tumor subpopulation lines that differ in metastatic phenotype. Although there was almost a 9-fold difference in spontaneous rates to ouabain resistance among the three lines tested, the difference did not correlate with ability to metastasize. Similarly a 10-fold difference in spontaneous rates to 6-thioguanine resistance did not correlate with metastatic ability. In contrast, the frequency of ethyl methanesulfonate-induced mutations was associated with metastatic potential. Thus, ethyl methanesulfonate only induced significant numbers of 6-thioguanine resistant colonies in 66 and 410.4 cells, the only 2 of 5 lines tested that spontaneously metastasize at high frequency, and of ouabain resistant colonies in 66, 410.4, and 168 cells, the only lines tested that produce experimental lung metastases after i.v. injection. Differential sensitivity to induced mutation was not correlated with differences in plating efficiency, wild type sensitivity to ethyl methanesulfonate, 6-thioguanine, or ouabain toxicity, ploidy, cell shape, cell size, or ability to engage in metabolic cooperation.

Chromosome and cell surface marker studies in 1-propyl-1-nitrosourea-induced thymic lymphomas of the rat.

Immunological cell surface markers were studied in seven transplantable 1-propyl-1-nitrosourea-induced thymic lymphoma lines in F344 rats by reactivity to anti-Thy-1.1, anti-rat Ig (anti-Ig), and anti-rat T-cell (anti-T) sera, and by the capacity to form rosettes with guinea pig red blood cells. All the tumor lines were estimated to be sensitive to anti-Thy-1.1 but insensitive to anti-Ig serum in the presence of complement. The differences in reactivity to anti-T serum and rosette-forming capacity (RFC) allowed classification of the lines into three types. In type I, three lines were highly sensitive to anti-T serum but low in RFC, indicating that these lymphomas probably originated from relatively mature intrathymic T cells. In type II, two lines were moderately sensitive to anti-T serum and relatively high in RFC, indicating that these lymphomas derived from intrathymic T cells. In type III lymphomas, the remaining two lines were not only insensitive to anti-T serum but also low in RFC, suggesting that these lymphomas might have arisen from immature precursors of T and/or B cells. The chromosome study revealed that type I lymphomas were diploid, with slight numerical and structural variations. Type II lymphomas were pseudodiploid or hypotetraploid, with considerable variation in the number and morphology of chromosomes. Type III lymphomas had a diploid or hyperdiploid constitution, with a moderate degree of karyotypic variation. Neither consistent nor common karyotypic alterations among the seven lines were found, although the karyotypic instability seemed to be related to the immunological types of the lymphoma lines, possibly reflecting the differentiation process of the target cells involved in the malignant transformation.

[Clinical evaluation of cefoxitin against intractable bronchopulmonary infections. Patients not responding to other conventional antibiotics (author's transl)].

Cefoxitin was administered to a total of 17 patients with intractable bronchopulmonary infections who had failed to respond to other conventional antibiotics, and the following results were obtained. (1) The clinical response, except 1 patient classified as evaluation impossible, was excellent in 3 patients, good in 10 and fair in 3 with an efficacy rate of 81.3%. (2) An improvement rate of more than 70% was observed in the findings of body temperature, dyspnea, colour of sputum, WBC and CRP. (3) There was no subjective nor objective side effects attributable to cefoxitin. In view of the results stated above, we have concluded that cefoxitin is a useful antibiotic for the treatment of intractable bronchopulmonary infections.