Cyclophosphamide-immunodepressed FVB/N mice: potentiating the effects of testicular cytomegalovirus infection.

The aim of this study was to investigate the effect of cyclophosphamide (CY) immunosuppression on the infection of germinal cells following testicular inoculation of male FVB/N mice with murine cytomegalovirus (MCMV). We used CY to modulate the immune status in order to mimic iatrogenic immunosuppression in humans (organ transplantation) as closely as possible. We show that viral pathological manifestations observed in mice treated with this CY-MCMV combination were severer than those observed in immunocompetent male mice infected with MCMV alone. As previously reported, the typical MCMV cellular inclusions were present in interstitial spaces; however, the spermatogenic cells were never directly infected. Nonetheless, at the end of our observation, we obtained definitive necrosis of the testes. These results suggest that germline cell necrosis induces sterility in immunodepressed infected male mice indirectly. In the case of organ transplantation, particular attention should be accorded to male patients receiving CY.

Failure to infect embryos after virus injection in mouse zygotes.

BACKGROUND: The intracytoplasmic injection of sperm raises the problem that viral elements may be transported into the oocyte by the spermatozoon or the surrounding medium. It also raises questions about how the developing zygote will behave. METHODS: We used the murine model to microinject murine cytomegalovirus (MCMV) into the zygote ooplasm and followed the changes in these microinjected zygotes in vivo and in vitro over time. RESULTS: 80% of zygotes microinjected with viral suspension, and 80% injected with medium alone, survived. Although MCMV DNA was detected in 56% of injected embryos, up until the blastocyst stage, the mice born from these injected zygotes developed normally and did not contain MCMV DNA. When embryonic stem cells were co-incubated with MCMV and then transferred into healthy blastocysts, the offspring were normal and did not contain any MCMV DNA. CONCLUSIONS: Our observations suggest that even if MCMV DNA persists from the zygote to the blastocyst stage, its presence has no detrimental effect on pre-implantation or post-implantation development.

Experimental inoculation of male mice with murine cytomegalovirus and effect on offspring.

BACKGROUND: Murine cytomegalovirus (MCMV) was used to examine aspects of viral infection in male mice, and its possible transmission to their offspring. METHODS AND RESULTS: FVB/N mice inoculated intratesticularly with 5x10(5) plaque forming units (PFU) of MCMV, developed peritoneal haemorrhagic exudates, spleen hypertrophy and acute local infection. Infectiousness was detected until 15 days post-inoculation (D15 PI) in the genital organs, and virus DNA up to D35 PI. Testicular endothelial and Leydig cells were infected, and peritubular cells severely damaged. Spermatogenesis was affected, but neither germ cells nor Sertoli cells were infected. No virus was found in the epididymal epithelial cells. Viral DNA was detected in cells extracted from vas deferens samples until D15 PI. Neither infectious virus nor viral DNA were found in spermatozoa recovered from uterine fluid, fertilized oocytes, blastocysts, fetal tissues or newborn animals following the mating of infected males with uninfected females. CONCLUSIONS: MCMV harboured in the male genital organs was not transmitted to their offspring, even when mating occurred during the acute phase of CMV disease. Although the infection may have had an impact on spermatogenesis, fertility was not affected. These results do not support the hypothesis of conceptus MCMV infection by the fertilizing spermatozoon in natural conception.

Identification of pancreatic type I secreted phospholipase A2 in human epidermis and its determination by tape stripping.

Phospholipases A2 (PLA2) catalyse the release of fatty acids from the sn-2 position of phospholipids and have been suggested to play a key part in permeability barrier homeostasis. Using a sensitive and versatile fluorometric method, significant PLA2 activity has been detected in both human skin homogenates and tape strippings of stratum corneum. Based on various properties (resistance to heat and sulphuric acid treatment, neutral optimal pH, absolute requirement for millimolar calcium concentrations, inhibition by dithiothreitol and p-bromophenacyl bromide, and resistance to a trifluoromethyl ketone derivative of arachidonic acid, AACOCF3, a specific inhibitor of cytosolic PLA2), this enzyme was characterized as a secretory PLA2 (sPLA2). Immunohistochemistry revealed strong labelling of type I pancreatic sPLA2 at the stratum corneum-stratum granulosum junction, type II sPLA2 being undetectable. An increase in PLA2 activity in tape-stripped material from the deepest level of the stratum corneum was correlated with partial morphological disappearance of type I sPLA2 immunolabelling. Our data thus provide the first convincing evidence that pancreatic sPLA2 is significantly expressed in human epidermis, where it might participate in the accumulation of free fatty acids contributing to the permeability barrier. In addition, our method for determining PLA2 activity in easily available tape strippings should allow further clinical studies aimed to explore possible PLA2 abnormalities in various dermatoses.

Testicular wild-type p53 expression in transgenic mice induces spermiogenesis alterations ranging from differentiation defects to apoptosis.

While p53 is dispensable for development, an excess of p53 has dramatic consequences on the embryogenesis and on the cell differentiation. In an attempt to analyse in vivo the effects of p53 activity, we have generated transgenic mice expressing the wild-type p53 under the control of the metallothionein I promoter. In the three transgenic lines established, exogenous p53 is expressed constitutively in the postmeiotic cells of transgenic males and two lines are subfertile. Transgenic males expressing the upper level of p53 produce few spermatozoa since the majority of developing spermatids undergo apoptosis. In the subfertile males exhibiting an intermediate amount of p53, teratozoospermia is obvious suggesting an altered terminal differentiation of postmeiotic cells. In contrast lower level of p53 does not lead the third line to sterility. These results suggest that the activity of p53 is dependent in vivo on the amount of p53 present within cells, as it has been already demonstrated in vitro.

Randomized trial of CVPP for three versus six cycles in favorable-prognosis and CVPP versus AOPE plus radiotherapy in intermediate-prognosis untreated Hodgkin's disease.

PURPOSE: To evaluate in a randomized trial the impact of three versus six cycles of cyclophosphamide, vinblastine, procarbazine, and prednisone (CVPP) chemotherapy in favorable-prognosis and CVPP versus doxorubicin, vincristine, prednisone, and etoposide (AOPE) plus involved-field radiotherapy (RT) in intermediate-prognosis previously untreated Hodgkin's disease. PATIENTS AND METHODS: Of 256 patients evaluated, 80 with a favorable prognosis according to a prognostic index designed by the Grupo Argentina de Tratamiento de Leucemia Aguda (GATLA) were randomized to three versus six cycles of CVPP without RT and 176 with intermediate risk to CVPP versus AOPE, both for six cycles with RT between the third and fourth cycles of 30 Gy to the involved areas at diagnosis. CVPP consisted of intravenous (I.V.) cyclophosphamide and vinblastine on days 1 and 8, and oral procarbazine and prednisone on days 1 to 14, every 28 days. AOPE consisted of I.V. doxorubicin and vincristine on day 1, oral prednisone on days 1 to 5, and I.V. etoposide on days 1 and 3, every 28 days. RESULTS: Complete remission was obtained in 39 of 41 (95%) patients treated with three cycles of CVPP and 36 of 39 (92%) treated with six cycles in the favorable-risk group (difference not significant [NS]). In the intermediate-risk group, 89 of 92 (97%) treated with CVPP plus RT versus 75 of 84 (89%) treated with AOPE plus RT achieved a complete remission (P = .05). At 60 months, the event-free survival (EFS) and overall survival rates in the favorable-risk group were 80% and 91% for CVPP x 3 and 84% and 97% for CVPP x 6, respectively (P = NS). In the intermediate-risk group, 60-month EFS rate for CVPP plus RT was 85%, compared with 66% for AOPE plus RT (P = .009). The overall survival rate was 95% versus 87% respectively (P = .157). CONCLUSION: Three cycles of CVPP without RT are equally effective as six cycles in the favorable-risk group. However, in the intermediate-group, CVPP plus RT is superior to AOPE plus RT, with significantly fewer events before and after induction (P = .009), without a difference in overall survival.

Multivariate analyses of prognostic factors associated with hematopoietic recovery in autograft patients with different sources of progenitor cells. A GATMO experience. Argintine Group of Bone Marrow Transplant.

OBJECTIVE: To evaluate in a multivariate analysis the prognostic factors associated with hematopoietic recovery and the supportive care requirements after autotransplant of progenitor cells (PC) from various sources: bone marrow (BMPC), BMPC & peripheral blood (PBPC), and PBPC alone. PATIENTS AND METHODS: A total of 570 patients with hematological malignancies and solid tumors underwent high-dose therapy followed by autotransplant. PBPC were obtained after mobilization with chemotherapy and/or cytokines. One-hundred five patients received BMPC, 217 received BMPC & PBPC and 248 PBPC alone; all of the patients received G-CSF or GM-CSF after infusion. RESULTS: In a multivariate analysis the recovery of neutrophils was adversely associated with low numbers of nucleated cells infused (P < 0.13), bone marrow progenitor cell source, and diagnosis of multiple myeloma and acute leukemia (P < 0.001). The factors that adversely affected platelet recovery were low number of nucleated cells and diagnosis of multiple myeloma and acute leukemia (P < 0.001). CONCLUSIONS: We conclude that BMPC adversely affect neutrophil recovery while low numbers of nucleated cells and diagnosis of multiple myeloma and acute leukemia adversely affect both neutrophil and platelet recovery.

Developmental control of IFN-alpha expression in murine embryos.

The expression of IFN-alpha transcripts was investigated in murine embryos, fetuses, and fetal annexes in mid and late pregnancy. We have shown by Northern blot analysis, reverse transcription-polymerase chain reaction, and in situ hybridization the presence of IFN-alpha transcripts in mouse placenta, fetus, and newborn. From the 14th day of gestation until birth, a typical IFN-alpha transcript (1.2 kb) is found in the fetus. A transcript of larger size (2.2 kb) appears near birth and is present in the newborn mouse. Fetal annexes between the 10th and 21st days of gestation also express IFN-alpha. From the 10th day until birth, the 1.2-kb IFN-alpha mRNA species is present, as well as unusually large transcripts: 4 and 7 kb. To localize IFN-alpha transcripts, in situ hybridization was performed, using 35S-IFN-alpha antisense RNA probe in comparison with the sense RNA probe. The tissue pattern of IFN-alpha transcription in fetuses shows a clear labeling of many epithelia, such as skin, ependyme, and intestine glandular epithelium. A possible relation with cellular differentiation is discussed.

Results of treatment with an intensive combination induction regimen containing idarubicin in children with acute myeloblastic leukemia: preliminary report of the Argentine Group for Treatment of Acute Leukemia.

In April 1990, the Argentine Group for Treatment of Acute Leukemia began a multicenter trial for the treatment of previously untreated acute myeloblastic leukemia patients who were under 21 years of age. Initial treatment consisted of an 8-day induction phase with cytarabine together with idarubicin on days 3 to 5 and etoposide on days 6 to 8. A multidrug consolidation phase was subsequently administered and, after a treatment-free interval of 2 to 4 weeks, two 5-day intensification courses with high-dose cytarabine and etoposide were delivered with a 4-week interval between each course. Continuation therapy was started 2 to 4 weeks after the second course, with 6-thioguanine daily and cytarabine daily for 4 days every 4 weeks. Treatment was stopped after 18 months in children in continuous complete remission. A preliminary evaluation of this ongoing study included 36 patients with a mean age of 7.5 years (age range, 5 months to 16 years). The majority of patients had a French-American-British classification of M2 (n = 13) or M4 (n = 8). Complete remission was achieved by 91.7% of patients, while one died from sepsis in bone marrow hypoplasia and two were regarded as treatment failures. At a median follow-up of 12 months (range, 2 to 23 months) there were 12 adverse events: six bone marrow relapses, one bone marrow/skin relapse, and five deaths in complete remission (all deaths occurred during the consolidation phase). During the induction phase most of the patients experienced prolonged myelosuppression, and grade 3 to 4 toxicity (according to the Children's Cancer Group criteria) was frequently seen. Alopecia was universal. However, toxicity was manageable. We conclude that idarubicin in combination with cytarabine and etoposide is a highly effective regimen for induction in children with acute myeloblastic leukemia.

[Comparative study of 1st generation versus 2d generation (CAVPE) combinations in intermediate and high-grade lymphomas]. / Estudio comparativo de combinaciones de primera generación versus segunda generación (CAVPE) en linfomas de grado intermedio y alto.

PURPOSE: To compare four first generation chemotherapy regimes (FGR) used by GATLA in low and intermediate grade lymphomas. Versus a second generation regimen called CAVPE. PATIENTS AND METHODS: A group of 205 patients treated with FGR (79 with BACOP, 89 with COPP, 19 with CHOP, and 18 with CNOP), and 244 others treated with the combination of cyclophosphamide, doxorubicin, vincristine, prednisone and etoposide (CAVPE) were included. Two randomized phase III multicentric studies, COPP vs BACOPP and CHOP vs CNOP, as a whole group, were compared with the second generation scheme, CAVPE. All the patients with FGR received 6 monthly treatment courses, and the CAVPE patients were given 8 monthly courses. RESULTS: The median age was 55 years (18-85) for the FGR group and 51 years (15-79) for the CAVPE group. The stage distribution for both FGR and CAVPE groups was, respectively, as follows: II: 38 and 55 cases; III 80 and 84 cases; IV: 87 and 105 cases. No significant differences were found between both groups when comparing other characteristics of the patients, namely, sex, symptoms, mediastinal, abdominal or extranodal involvement, liver, spleen or bone-marrow infiltration, and bulky tumoural mass. The percentage of complete remission (CR) was 52% (107/205) in the patients treated with FGR, and 67% (163/244) in the CAVPE group (p < 0.001). The estimated probability of sustained first CR at 72 months was 38% for the FGR group and 54% for the CAVPE group (p = 0.0167), whereas 17% and 36%, respectively, of the evaluable patients were alive and well, with no signs of disease progression (p = 0.000). The 72-month survival was estimated in 32% and 51%, respectively, for each group (p = 0.0004). CONCLUSIONS: 1. CAVPE seems to offer better responses and disease-free survival in this non-selected group of low and intermediate lymphoma patients. 2. The better results in terms of CR and lower incidence of relapses attained with CAVPE with regard to FGR are probably related with a number of facts, such as the use of more aggressive drugs, higher doses, a better drug combination rationale, the inclusion of new drugs, such as etoposide, or a selection of the patients.

Enhancement by stable butyrate derivatives of antitumor and antiviral actions of interferon.

The use of n-butyric acid has been associated with induction of cell differentiation and bypassing of genetic defects in the suppression of malignancy. This biological response modifier satisfies the requirements for specificity and low toxicity, and its use can be considered as an alternative approach to conventional cancer chemotherapy. However, a lack of clinical efficacy has been observed with butyrate and attributed mainly to the rapid metabolism of the compound. Butyric acid pro-drugs derived from monosaccharides such as 3-O-butanoyl-1,2-O-isopropylidene-alpha-D-glucofuranose (MAG = 3but) have consequently been devised. Pharmacokinetic and biological advantages of MAG = 3but have been previously described. In the present report, we have studied the effect of MAG = 3but on murine interferon-alpha, beta (IFN) anticellular, antitumor and antiviral activities. In vitro, it appears that MAG = 3but predisposes malignant MSV cells to a later, complete establishment of the antiproliferative and the cell-differentiating effects of IFN, and the antiviral action of the latter in the same line of cells infected with encephalomyocarditis (EMC) virus. In vivo, combined treatment with MAG = 3but and IFN protects mice effectively against the fatal development of ascitic sarcoma 180 TG and the lethal effect of EMC virus infection.

Pharmacokinetic studies of N-butyric acid mono- and polyesters derived from monosaccharides.

The pharmacokinetics of seven butyric esters derived from monosaccharides were studied after iv administration of a bolus dose to rabbits. Results obtained showed that a constant plasma level of butyric acid is maintained due to the slow disappearance of butyric acid esters from the plasma in contrast to the case of salts, such as arginine butyrate, which are rapidly cleared. The maintenance of these covalent compounds in the body can increase concentrations of n-butyric acid in the tumor area for more efficient chemotherapy. These results seem to be directly related to the in vitro anticellular activity of butyric esters and the prolonged therapeutic protection in tumor-bearing animals.

[Protective activity of monoacetone glucose 3-butyrate, prodrug of n-butyric acid, against the fatal effect of encephalomyocarditis virus in mice]. / Action protectrice du butyrate-3 monoacétone glucose, prodrogue de l'acide n-butyrique, contre l'effet létal du virus de l'encéphalomyocardite de la souris.

A comparative study was made, in the mouse, on antiviral properties of a prodrug of n-butyric acid derived from monosaccharides: monoacetone glucose 3-butyrate (MAG = 3but), and of a free form of n-butyric acid: arginine butyrate (BuO Arg). Preventive injection of MAG = 3but protected the mice twice as effectively as BuO Arg against the lethal effect of 100 LD50 of encephalomyocarditis virus. Under the same experimental conditions, monoacetone glucose (MAG) used as carrier of the biologically active moiety, was inactive on its own. Antiviral activity of MAG = 3but was shown not to be virucidal, but rather could involve stimulation of the immune system. This capability supplements known anticellular and antitumoral properties. In total these results indicate a prime therapeutic importance for this new molecule, pharmacokinetically suitable, with its very low toxicity, for clinical application. Combined use of MAG = 3but with biological response modifiers which have similar affinity, such as Interferon, is discussed.

Butyric monosaccharide ester-induced cell differentiation and anti-tumor activity in mice. Importance of their prolonged biological effect for clinical applications in cancer therapy.

The interest of butyric salts is based on their capacity to promote differentiation of malignant cells and inhibition of tumor development. The phenotypic modifications are rapidly reversible and require the continuous presence of butyric salts in the target area, which raises problems for therapeutic applications. We show here that the covalent binding of n-butyric acid on natural polyhydroxylated compounds such as monosaccharides, especially 3- or 6-O-butanoyl-1,2-O-isopropylidene-alpha-D-glucofuranose, retains the majority of the biological properties of n-butyric acid. The delayed degradation of these covalent compounds is associated with an improved maintenance of cell differentiation and anti-tumor protection in mice. These butyric complexes thus seem potentially useful for therapeutic applications.

Low level toxicity and antitumor activity of butyric mono- and polyester monosaccharide derivates in mice.

This study compares the antitumor activity of five mono- and polyesters of n-butyric acid derived from monosaccharides in the murine model of Crocker 180 TG Sarcoma. Tumor incidence at ten days, mean survival time and final survival rate were significantly affected in all cases. Combined treatment by butyric esters, alpha/beta interferon (IFN) and/or Corynebacterium parvum used as immunestimulator improved the antitumor protection. Studies of acute toxicity in mice, performed by i.p. and oral routes, showed the low toxicity of butyric esters, which were devoid of detectable side effects with no incidence on ponderal growth when administered per os in rats daily for one month. Finally, a comparative study of antitumor activity, toxicity and water-solubility of various butyric esters enabled us to select among these new molecules two isomers (carbon-3 and carbon-6 of the glucose ring substituted with n-butyric acid) derived from monoacetone glucose for further investigations of their biological mechanism in vivo and in vitro.

Anti-tumor protection induced in mice by fatty acid conjugates: alkyl butyrates and poly(ethylene glycol) dibutyrates.

Simple fatty acids, especially butyrate salts, have interesting biological properties, since they are able to down-regulate cell growth and promote various differentiated cellular functions. Their use for anti-tumor treatment is, however, hampered by their over-rapid diffusion in the blood, followed by a short-lived biological action. We have therefore devised conjugates linking butyrate with either (i) aliphatic alcohols of increasing carbon numbers ranging from C4 to C12 or (ii) poly(ethylene glycols) of increasing molecular weights. In both cases, the resulting butyric esters can be hydrolysed by esterases which can release biologically active subunits from the synthetic compounds. As shown in the present study, only one conjugate in each series gave satisfactory anti-tumor protection: namely, I-octyl butyrate and poly(ethylene glycol 1000) dibutyrate respectively. A single immune-stimulatory injection of purified Corynebacterium parvum extract prior to administration of the conjugates significantly increased the anti-tumor potency.

Pharmacokinetic study of butyric acid administered in vivo as sodium and arginine butyrate salts.

Considering that butyrate-treated malignant cells can recover in a transitory fashion a non-cancerous phenotype, the authors carried out a pharmacokinetics study of butyric acid injected as sodium or arginine salts for possible antitumor therapies. In the case of 1-14C-labelled butyrate, the appearance of radioactivity in the blood of injected mice is rapid and some of it is maintained for relatively long periods in different organs, mainly the liver. However, no precision can be given about the structure of radioactive compounds in blood and tissues. Using gas-liquid chromatography, the authors studied the metabolism of butyrate in both animals and man. In mice and rabbits, the half-life is less than 5 min. In man, the butyric acid elimination curve can be divided into two parts corresponding to two half-lives: for the first (0.5 min), the slope suggests an accelerated excretion, while for the following (13.7 min), a slow plateau is observed. The rapid elimination of butyrate is a limiting factor for practical applications. However, the lack of toxicity supports its use in human therapy.

Detection of vesicular stomatitis virus (VSV) RNA in the central nervous system of infected mice by in situ hybridization.

Using in situ hybridization with a cloned DNA probe specific for the VSV G protein, viral RNA was detected and localized in CNS tissue of mice infected i.c. with either wild or ts G 31 VSV mutant. In both cases, brain and spinal cord neurons were the only cells seen to contain viral RNA. Virus-positive neurons were observed enclosed in spongious areas induced by the ts VSV mutant. These results suggest that the VSV shows a strong tropism for the neuronal cell and indicate that the vacuole formation might be associated with the expression of the VSV G protein gene in infected neurons.

Immunomodulatory effects of arginine butyrate.

We have studied the effect of arginine butyrate on T cell and macrophage functions. When target cells are treated with this substance, they become resistant to T cell-mediated cytotoxicity, as detected by the chromium assay. In contrast, when effector T cells are treated, the cytotoxicity seems to be augmented. Peritoneal macrophages incubated with butyrate are increasingly adhesive to substrate. After in vivo treatment, spleen derived macrophages show an augmented cytostatic capacity in the presence of L1210 cells and an enhanced phagocytic activity for IgG-coated erythrocytes. To sum up, the overall effects of butyrate salts on different immune functions are somewhat reminiscent of that of interferon. It is likely that these immune effects contribute, at least in part, to explain its antitumor properties observed in grafted tumors in mice.