The impact of overweight on lipid phenotype in different forms of dyslipidemia: a retrospective cohort study.

PURPOSE: Dyslipidemia plays a pivotal role in increasing cardiovascular risk. In clinical practice the misleading association between altered lipid profile and obesity is common, therefore genetically inherited dyslipidemias may not completely be addressed among patients with overweight. Thus, we aim to investigate the influence of overweight and obesity on the lipid phenotype in a cohort of patients with different forms of dyslipidemia. METHODS: A retrospective analysis was conducted on patients with dyslipidemia from 2015 to 2022. Patients were stratified in familial hypercholesterolemia (FH), familial combined hyperlipidemia (FCHL), non-familial hyperlipidemia or polygenic hypercholesterolemia (PH). Clinical characteristics and lipid profile were evaluated. RESULTS: Of the total of 798 patients, 361 were affected by non-familial hyperlipidemia (45.2%), while FCHL, FH and PH was described in 19.9%, 14.0% and 20.9% of patients, respectively. Overweight prevalence was higher in FCHL and non-familial hyperlipidemia patients than FH and PH patients. Subjects with overweight and obesity were independently associated with lower levels of high-density lipoprotein cholesterol (HDL-C) compared to patients with normal weight (52.4 and 46.0 vs 58.1, respectively; p < 0.0001); levels of triglycerides (TG) and non-HDL-C were higher in patients with overweight and obesity than patients with normal weight (257.3 and 290.9 vs 194.8, and 221.5 and 219.6 vs 210.1, p < 0.0001 and p = 0.01, respectively), while no differences were observed between patients with overweight and obesity. CONCLUSION: While dyslipidemias can be influenced by various factors, an important determinant may lie in genetics, frequently acting as an underlying cause of altered lipid profiles, even in cases of overweight conditions.

The combined therapy with myo-inositol and D-chiro-inositol reduces the risk of metabolic disease in PCOS overweight patients compared to myo-inositol supplementation alone.

BACKGROUND: PCOS is the main cause of infertility due to metabolic, hormonal and ovarian dysfunctions. Women affected by PCOS often suffer of insulin resistance and of a compensatory hyperinsulinemia. These conditions put the patients at risk of developing several metabolic disorders. Both myo-inositol (MI) and D-chiro inositol (DCI) glycans administration has been reported to exert beneficial effects at metabolic, hormonal and ovarian level. Beside these common features, MI and DCI are indeed different molecules: they belong to two different signal cascades and regulate different biological processes. AIM: In this study, we aim to verify whether the two molecules have a synergistic action by acting on their specific cellular pathways. The effectiveness in reducing the risk of metabolic syndrome as well as in enhancing the ovarian functions of a combined therapy with MI and DCI was compared to a mono therapy in a randomized controlled trial. METHODS: Fifty overweight women with PCOS were enrolled and divided in two groups to receive MI and DCL (MI+DCI group) or MI alone (MI group) for a period of six months. Baseline measurements were repeated at three months (T1) and at the end of the treatment (T2). RESULTS: At the end of the treatment, both MI and MI+DCI groups showed an improvement of the metabolic parameters and no significant differences were found. As expected, the combined supplementation with MI and DCI resulted to be more effective, compared to the MI group, after three months of treatment. CONCLUSIONS: The combined administration of MI and DCI in physiological plasma ratio (40:1) should be considered as the first line approach in PCOS overweight patients, being able to reduce the metabolic and clinical alteration of PCOS and, therefore, reduce the risk of metabolic syndrome.

Normative data for lung function and exhaled nitric oxide in unsedated healthy infants.

Despite association with lung growth and long-term respiratory morbidity, there is a lack of normative lung function data for unsedated infants conforming to latest European Respiratory Society/American Thoracic Society standards. Lung function was measured using an ultrasonic flow meter in 342 unsedated, healthy, term-born infants at a mean ± sd age of 5.1 ± 0.8 weeks during natural sleep according to the latest standards. Tidal breathing flow-volume loops (TBFVL) and exhaled nitric oxide (eNO) measurements were obtained from 100 regular breaths. We aimed for three acceptable measurements for multiple-breath washout and 5-10 acceptable interruption resistance (R(int)) measurements. Acceptable measurements were obtained in ≤ 285 infants with high variability. Mean values were 7.48 mL·kg⁻¹ (95% limits of agreement 4.95-10.0 mL·kg⁻¹) for tidal volume, 14.3 ppb (2.6-26.1 ppb) for eNO, 23.9 mL·kg⁻¹ (16.0-31.8 mL·kg⁻¹) for functional residual capacity, 6.75 (5.63-7.87) for lung clearance index and 3.78 kPa·s·L⁻¹ (1.14-6.42 kPa·s·L⁻¹) for R(int). In males, TBFVL outcomes were associated with anthropometric parameters and in females, with maternal smoking during pregnancy, maternal asthma and Caesarean section. This large normative data set in unsedated infants offers reference values for future research and particularly for studies where sedation may put infants at risk. Furthermore, it highlights the impact of maternal and environmental risk factors on neonatal lung function.

Combined interleukin 1/interleukin 2 therapy of mice injected with highly metastatic Friend leukemia cells: host antitumor mechanisms and marked effects on established metastases.

Peritumoral injection of recombinant human interleukin 1 beta (IL-1 beta) in mice transplanted subcutaneously with Friend erythroleukemia cells (FLC) resulted in a marked increase in survival time and inhibition of metastatic tumor growth in liver and spleen. In contract, IL-2 treatment alone did not significantly inhibit the development of FLC metastases. A synergistic antitumor effect was observed after combined IL-1/IL-2 therapy of these mice. The antitumor action of IL-1/IL-2 treatment was abolished or markedly reduced in mice treated with antibodies to CD4 or CD8 antigens, whereas antibodies to asialo-GM1 were ineffective. A clear-cut increase in the percentage of CD4+ cells was observed in the spleens of cytokine-treated mice on days 17 and 23. On day 23 of cytokine therapy, CD8+ cells were increased in both spleens and lymph nodes. On day 17, infiltrates of host-reactive cells (i.e., lymphocytes, granulocytes, and monocytes) were observed in both spleen and liver from FLC-injected mice treated with IL-1/IL-2, in association with tumor cells. On days 17 and 23, spleen cells and cells recovered from mesenteric lymph nodes of IL-1/IL-2-treated mice exerted a potent antitumor effect as determined by Winn assay experiments. This antitumor activity was abolished by preincubation of spleen cells with anti-CD8 antibody, but not by treatment with antibodies to asialo-GM1; antibodies to CD4 exerted only a slight effect. Combined IL-1/IL-2 therapy was more effective on established (i.e., 6-7-d) FLC tumors than on early (i.e., 1-d) tumor-transplanted mice. IL-1/IL-2 treatments were also highly effective in increasing survival time of mice from which the subcutaneous primary tumors were excised 7 d after FLC injection. These data indicate that in mice injected with FLC, the antitumor effects of IL-1/IL-2 are mediated by CD4+ and CD8+ cells (but not NK cells), and suggest that this combined cytokine treatment may be effective against established metastatic tumors.

Importance of cyclophosphamide-induced bystander effect on T cells for a successful tumor eradication in response to adoptive immunotherapy in mice.

Cyclophosphamide (CTX) increases the antitumor effectiveness of adoptive immunotherapy in mice, and combined immunotherapy regimens are now used in some clinical trials. However, the mechanisms underlying the synergistic antitumor responses are still unclear. The purpose of this study was (a) to evaluate the antitumor response to CTX and adoptive immunotherapy in mice bearing four different syngeneic tumors (two responsive in vivo to CTX and two resistant); and (b) to define the mechanism(s) of the CTX-immunotherapy synergism. Tumor-bearing DBA/2 mice were treated with a single injection of CTX followed by an intravenous infusion of tumor-immune spleen cells. In all the four tumor models, a single CTX injection resulted in an impressive antitumor response to the subsequent injection of spleen cells from mice immunized with homologous tumor cells independently of the in vivo response to CTX alone. Detailed analysis of the antitumor mechanisms in mice transplanted with metastatic Friend leukemia cells revealed that (a) the effectiveness of this combined therapy was dependent neither on the CTX-induced reduction of tumor burden nor on CTX-induced inhibition of some putative tumor-induced suppressor cells; (b) the CTX/immune cells' regimen strongly protected the mice from subsequent injection of FLC, provided the animals were also preinoculated with inactivated homologous tumor together with the immune spleen cells; (c) CD4(+) T immune lymphocytes were the major cell type responsible for the antitumor activity; (d) the combined therapy was ineffective in mice treated with antiasialo-GM1 or anti-IFN-alpha/beta antibodies; (e) spleen and/ or bone marrow cells from CTX-treated mice produced soluble factors that assisted in proliferation of the spleen cells. Altogether, these results indicate that CTX acts via bystander effects, possibly through production of T cell growth factors occurring during the rebound events after drug administration, which may sustain the proliferation, survival, and activity of the transferred immune T lymphocytes. Thus, our findings indicate the need for reappraisal of the mechanisms underlying the synergistic effects of CTX and adoptive immunotherapy, and may provide new insights into the definition of new and more effective strategies with chemotherapy and adoptive immunotherapy for cancer patients.

Combined interleukin-1 beta/interleukin-6 treatment in mice: synergistic myelostimulatory activity and myelorestorative effect after cyclophosphamide-induced myelosuppression.

We have studied the effects of single and combined treatment with recombinant human interleukin 1 beta (IL-1 beta) and recombinant human interleukin-6 (IL-6) on spleen and bone marrow hematopoiesis in normal and cyclophosphamide-treated mice. Injection of IL-1 beta alone resulted in a significant increase in the number of granulocytes and splenic progenitors [burst-forming units-erythroid (BFU-E) and colony-forming units-granulomonocytic (CFU-GM)] as compared with control mice but did not markedly enhance the number of bone marrow BFU-E and CFU-GM. IL-6 alone had little effect on the number of splenic progenitors but significantly increased the number of marrow BFU-E and CFU-GM, especially after a 6-day cytokine treatment. Combined daily administration of IL-1 beta and IL-6 for 3 days resulted in a synergistic stimulation of hematopoiesis as evaluated by the number of spleen and bone marrow CFU-GM and BFU-E colonies. Likewise, IL-1 beta/IL-6 markedly enhanced the number of circulating neutrophils, whereas each cytokine alone had little or no effect. When the numbers of spleen progenitors and peripheral granulocytes were determined 1 day after the last injection, a synergistic myelostimulatory effect of combined IL-1 beta/IL-6 treatment was observed at all doses (IL-1 beta, 0.25-0.5 microgram; IL-6, 1-20 micrograms). Furthermore, combined treatment with IL-1 beta/IL-6 accelerated and potentiated the recovery of myeloid cells after cyclophosphamide injection, whereas the single regimen treatment was not effective. Particularly, the rebound of WBC (especially neutrophilic granulocytes) after cyclophosphamide treatment was markedly enhanced by the combined treatment, whereas the single regimen was ineffective. Altogether these results may contribute to the development of combination therapies with cytokines and antiblastic agents in the treatment of cancer patients.

Inhibition of mouse alpha/beta-interferon of the multiplication of alpha/beta-interferon-resistant Friend erythroleukemia cells cocultured with mouse hepatocytes.

Administration of alpha/beta-interferon (IFN) exerts a marked antitumor effect in DBA/2 mice given injections i.v. of large numbers of IFN-alpha/beta-resistant erythroleukemia cells (FLC). To investigate the possible mechanisms of FLC tumor inhibition in the liver of interferon-treated mice, we developed an in vitro model consisting of a coculture of IFN-alpha/beta-resistant 3Cl8 FLC and syngeneic mouse hepatocytes. Whereas IFN-alpha/beta did not inhibit the multiplication of 3Cl8 FLC cultivated alone, it effectively inhibited the multiplication of 3Cl8 FLC in coculture with hepatocytes. The inhibitory effect was directly proportional to the amount of IFN-alpha/beta added to the cocultures, and more than 90% inhibition of FLC multiplication was noted with 1.6 x 10(5) IU/ml of IFN-alpha/beta on Day 3 of coculture. When FLC were separated from the monolayer of hepatocytes by a pored membrane (0.4 microns), the inhibitory effect on FLC proliferation was unchanged, indicating that a soluble factor(s) released from IFN-treated hepatocytes was most important in the inhibition of FLC multiplication. An inhibitory activity of FLC multiplication was detected only in the conditioned medium of IFN-treated hepatocytes but not in the conditioned medium of control hepatocytes nor in extracts of IFN-treated or control hepatocytes. The inhibitory factor(s) in the conditioned medium of IFN-treated hepatocytes was retained by an ultrafiltration membrane (Mr cut off 10,000), and its activity was completely abrogated by trypsin digestion. Its stability to treatment with 1 M acetic acid as well as lack of correlation between the antiproliferative effect and the amount of L-arginine in the medium distinguished this factor(s) from liver arginase which was also found to be a potent inhibitor of FLC multiplication in vitro. The inhibitory factor(s) was also distinguishable in its biological activity from IFN gamma, interleukin 1 alpha and beta, and transforming growth factor beta 1 and beta 2. These results suggest the possibility that the inhibitory effect of IFN-alpha/beta on the development of 3Cl8 FLC in the livers of IFN-treated mice may be mediated by an IFN-induced inhibitor of FLC multiplication.

Nuclear magnetic resonance analysis of tumor necrosis factor-induced alterations of phospholipid metabolites and pH in Friend leukemia cell tumors and fibrosarcomas in mice.

The alterations induced on the pool sizes of five phospholipid metabolites, glycerol 3-phosphorycholine, glycerol 3-phosphorylethanolamine, phosphorylcholine, sn-glycerol 3-phosphate, and choline were studied by nuclear magnetic resonance (NMR) spectroscopy in murine tumors injected with recombinant murine tumor necrosis factor (TNF). Solid tumors were obtained by s.c. injection of either Friend leukemia cells (clones 3C1-8 and 745) in DBA/2 mice or murine fibrosarcoma cells (HeN4) in C3H/HeN mice. After tumor nodules had developed, TNF or bovine serum albumin was injected intratumorally. Treatment of both tumors with TNF resulted in a marked inhibition of tumor growth. 31P-NMR analyses of Friend leukemia cell tumors (and tissue extracts), 6 h after injection of TNF, showed: (a) a 1.5- to 3.5-fold decrease in the pool sizes of glycerol 3-phosphorylcholine and glycerol 3-phosphorylethanolamine; (b) a 7- to 8-fold increase of sn-glycerol 3-phosphate; (c) a 2- to 3.5-fold decrease of phosphorylcholine; (d) an alkaline shift (0.2 units) in intratumoral pH. Similar metabolic alterations occurred in TNF-treated HeN4 fibrosarcoma. 1H-NMR analyses of Friend leukemia cell tumor extracts also indicated, 6 h after tumor injection with TNF: (a) elevated choline levels (9X); (b) a 19-fold increase in the ratio [choline]/[phosporylcholine]; (c) elevated (1.4X) levels of lactic acid; and (d) a 1.6-fold decrease in the [taurine]/[glycine] ratio. The results are interpreted in the light of possible alterations in the activity of enzymes controlling the de novo biosynthesis and catabolism of phospholipids. We concluded that NMR spectroscopy can be a useful means to monitor the level of some phospholipid precursors and/or derivatives as early markers of therapeutic efficacy in intact neoplastic tissues.

31P-nuclear magnetic resonance analysis of interferon-induced alterations of phospholipid metabolites in interferon-sensitive and interferon-resistant Friend leukemia cell tumors in mice.

Adult DBA/2 mice were given injections s.c. with either interferon-sensitive (745) or -resistant (3Cl-8) Friend erythroleukemia cells (FLC). After tumor nodules had developed, mouse interferon-alpha/beta was injected daily into the tumor. 31P-Nuclear magnetic resonance (NMR) spectroscopy examinations were undertaken on freshly dissected tumors at different days of treatment with either interferon or control preparations. Analysis of 745 FLC tumors in untreated mice at different days of tumor growth (day 8 to 13 after tumor implantation) showed marked increases in the levels of phosphorylcholine (PCho), glycerophosphorylethanolamine (GroPEtn) and glycerophosphorylcholine (GroPCho). In contrast high levels of PCho, GroPEtn and GroPCho were already detectable in the 3Cl-8 FLC tumors on day 8, and no significant changes were observed during subsequent tumor growth. The intracellular pH value remained practically constant in both FLC tumors. Daily intratumoral administration of either partially purified (10(7) IU/mg of protein) or highly purified (10(9) IU/mg of protein) mouse interferon-alpha/beta to both cell tumors resulted in decreases in the levels of PCho, GroPEtn and GroPCho and in increases in the intracellular pH with respect to tumors treated with control preparations or left untreated. Two days of daily treatment of mice with interferon sufficed to induce these metabolic changes which preceded the appearance of necrosis in the tumors. Treatment of FLC tumors with X-rays on day 12 of tumor growth did not result in any comparable metabolic changes 2 days after irradiation. Changes in the levels of phospholipid metabolites were not observed when 745 or 3Cl-8 cells were cultivated in the presence of interferon. As interferon induced these changes in both interferon-sensitive and -resistant tumors we conclude that interferon treatment results in host-mediated effects on the biosynthesis and/or catabolism of tumor cell phospholipids.

Alpha 1-interferon gene transfer into metastatic Friend leukemia cells abrogated tumorigenicity in immunocompetent mice: antitumor therapy by means of interferon-producing cells.

Highly metastatic alpha/beta-interferon (IFN-alpha/beta)-resistant Friend leukemia cells (FLC) were transfected with a retroviral vector (pLTneoL-5) containing the mouse IFN-alpha 1 gene. Transfected clones were isolated and tested for their capacity to secrete IFN-alpha 1 and their tumorigenicity when injected s.c. into immunocompetent syngeneic DBA/2 mice. Almost all FLC clones producing IFN in the range of 16-512 units/ml failed to grow when injected s.c. or i.p. into normal mice, whereas control FLC (transfected with a vector without the IFN gene) exhibited the highly malignant phenotype of the original FLC. High levels of IFN were detected in peritoneal fluid, tumor extracts, and sera of mice given injections of IFN-producing cells. Injection of mice with antibodies to IFN-alpha/beta resulted in the development of tumor ascites in mice transplanted i.p. with IFN-producing FLC. In contrast to the tumor rejection observed in immunocompetent mice, IFN-producing FLC were highly tumorigenic when transplanted into immunosuppressed nude mice. Mice given injections of IFN-producing FLC developed a long-lasting tumor-specific immune resistance to subsequent injection with highly metastatic FLC. Simultaneous s.c. injection of both metastatic FLC (approximately 10(3) 50% lethal doses) and IFN-producing cells resulted in potent inhibition of the tumor growth, with a survival rate of approximately 50% for injected mice. Contralateral injection (s.c.) of IFN-producing FLC into mice with established metastatic tumors produced a marked inhibition of tumor growth, with a survival rate of 10% for injected mice. These results indicate that: (a) the genetic modification of highly metastatic FLC by means of transfer of the IFN-alpha 1 gene results in potent tumor cell rejection, which is mediated by an IFN-induced host immune response; (b) injections of IFN-producing tumor cells are effective in inhibiting tumor growth in mice with established metastatic tumors. These data suggest that tumor cells transfected with the IFN-alpha gene might be used as an effective therapy for the treatment of certain human metastatic tumors, provided that suitable strategies are defined to prevent growth of the cytokine-producing cells.

Combined interleukin 1 beta/interleukin 2 treatment in mice: synergistic myelostimulatory activity and protection against cyclophosphamide-induced myelosuppression.

We have studied the effects of single and combined treatment with interleukin 1 beta (IL-1 beta) and interleukin 2 (IL-2) on spleen and bone marrow hematopoiesis in normal mice. Injection of IL-1 beta alone was followed by a significant increase in the number of granulocytes in spleen and progenitors (burst-forming units-erythroid and colony-forming units-granulomonocytic) in both spleen and bone marrow, s compared to control mice. In contrast, IL-2 alone induced only a slight increase in the number of marrow colony-forming units-granulomonocytic and had no significant effect on spleen progenitors. Repeated injections of both IL-1 beta and IL-2 resulted in a synergistic increase in spleen weight and splenocyte number, as compared to mice treated with the single cytokine regimen; in particular, the combined treatment induced a marked rise in the number of neutrophilic granulocytes and erythroblasts, whereas splenic lymphocytes were not affected. This regimen also caused a synergistic increase in the number of spleen and marrow progenitor cells: a time-course analysis showed an elevation in numbers of both burst-forming units-erythroid and colony-forming units-granulomonocytic, first in marrow (day 10) and subsequently in spleen (day 18). Combined IL-1 beta/IL-2 treatment dampened the decrease and accelerated the recovery of myeloid cells after cyclophosphamide injection, whereas the single cytokine regimen was not effective. Similarly, the rebound of WBC (especially neutrophilic granulocytes) after cyclophosphamide treatment was markedly enhanced by the combined treatment, whereas the single cytokine regimen was ineffective. These results, indicating a myelostimulatory effect by the combined cytokine regimen, together with our previous observations showing a synergistic antitumor activity by IL-1/IL-2 treatment in experimental mouse tumors (V. Ciolli et al., J. Exp. Med., 173: 313-322, 1991), may provide the basis for the development of new combination therapies with cytokines and antiblastic agents in the treatment of cancer patients.

T-cell-directed TAL-1 expression induces T-cell malignancies in transgenic mice.

The TAL-1 gene specifies for a basic domain-helix-loop-helix protein, which is involved in the control of normal hematopoiesis. In human pathology, the TAL-1 gene product is expressed in a high percentage of T-cell acute lymphoblastic leukemias in the pediatric age range; however, it has not been established whether the expression has a causal role in oncogenesis. In this report, we describe the phenotype of mouse transgenic lines obtained by inducing tal-1 protein expression in lymphoid tissues using the LCK promoter. The survival rate of tal-1 transgenic animals was much lower as compared with control mice. Histopathological analysis revealed lymphomas of T-cell type, often comprising a minor B-cell component. Some mice showed marked splenic lymphocyte depletion. Primary lymphocyte cultures showed partial independence from exogenous growth stimuli and increased resistance to low-serum apoptosis. To further unravel the tal-1 oncogenic potential, a strain of tal-1 transgenic mice was crossbred with p53-/- mice; the survival rate in these animals was reduced by more than one-half when compared with that of tal-1 mice, and histopathological analysis revealed exclusively T-cell lymphomas. These data indicate that TAL-1, expressed in T cells, is per se a potent oncogene, which may exert a key leukemogenetic role in the majority of T-cell acute lymphoblastic leukemias.

The induction of in vivo proliferation of long-lived CD44hi CD8+ T cells after the injection of tumor cells expressing IFN-alpha1 into syngeneic mice.

The tumorigenicity of transplantable tumor cells in mice is reduced by transduction with cytokine genes, including IFN-alpha and interleukin (IL) 12. Although T cells are considered important in tumor rejection, the mechanism by which genetically modified tumor cells stimulate the immune system has not been examined. In this study, the in vivo proliferation of T-cell subsets in mice transplanted with cytokine-producing syngeneic tumor cells was assessed by administering the DNA precursor bromodeoxyuridine. The injection of viable cells producing IFN-alpha or IL-12 caused a marked proliferation of CD8+ T lymphocytes in both the spleen and lymph nodes. Proliferation was most prominent among memory-phenotype CD44hi CD8+ T cells. In contrast, proliferation of CD8+ T cells did not occur in mice injected with control cells or with cells expressing IL-4, granulocyte colony-stimulating factor, or IFN-gamma. Pulse-chase studies in mice injected with IFN-alpha-producing cells showed that a proportion of proliferating CD8+ T cells survived for at least 70 days, suggesting that long-lived memory cells are induced using such an approach. In summary, these results, together with previous studies on the host immune reactivity triggered by the injection of tumor cells expressing IFN-alpha, represent a strong rationale for considering IFN-alpha as a powerful T-cell adjuvant for the generation of more effective cancer vaccines.

Primary HIV-1 infection of human CD4+ T cells passaged into SCID mice leads to selection of chronically infected cells through a massive fas-mediated autocrine suicide of uninfected cells.

We have recently shown that a human CD4+ T cell line (CEM-SS) acquires the permissiveness to M-tropic strains and primary isolates of HIV-1 after transplantation into SCID mice. This permissiveness was associated with the acquisition of a memory (CD45RO+) phenotype as well as of a functional CCR5 coreceptor. In this study, we have used this model for invest-igating in vivo the relationships between HIV-1 infection, apoptosis and T cell differentiation. When an in vivo HIV-1 infection was performed, the CEM cell tumors grew to a lower extent than the uninfected controls. CEM cells explanted from uninfected SCID mice (ex vivo CEM) underwent a significant level of spontaneous apoptosis and proved to be CD45RO+, Fas+ and Fas-L+, while Bcl-2 expression was significantly reduced as compared to the parental cells. Acute HIV-1 infection markedly increased apoptosis of uninfected ex vivo CEM cells, through a Fas/Fas-L-mediated autocrine suicide/fratricide, while parental cells did not undergo apoptosis following viral infection. The susceptibility to apoptosis of ex vivo CEM cells infected with the NSI strain of HIV-1, was progressively lost during culture, in parallel with the loss of Fas-L and marked changes in the Bcl-2 cellular distribution. On the whole, these results are strongly reminiscent of a series of events possibly occurring during HIV-1 infection. After an initial depletion of bystander CD4+ memory T cells during acute infection, latently or chronically infected CD4+ T lymphocytes are progressively selected and are protected against spontaneous apoptosis through the development of an efficient survival program. Studies with human cells passaged into SCID mice may offer new opportunities for an in vivo investigation of the mechanisms involved in HIV-1 infection and CD4+ T cell depletion.

Vaginal transmission of HIV-1 in hu-SCID mice: a new model for the evaluation of vaginal microbicides.

OBJECTIVE: To develop an animal model of vaginal transmission of HIV-1 for the evaluation of vaginal microbicides. DESIGN: Vaginal infection was performed in SCID mice reconstituted with 4 x 107 human peripheral blood lymphocytes (hu-PBL) by non-invasive vaginal administration. The hu-PBL were previously infected in vitro with a non-syncytium (NSI) strain of HIV-1 (SF162) (hu-PBL-SCID). Lymphocyte migration in vivo was examined using fluorescently labelled human lymphocytes. METHODS: The percentage of CD4 T cells, plasma viral load and p24 antigen were evaluated using fluorescent activated cell sorting (FACS), the Amplicor HIV-1 monitor kit and enzyme-linked immunosorbent assay, respectively. Polymerase chain reaction (PCR) analysis was performed on DNA extracted from spleen and lymph nodes. For in vivo migration of labelled lymphocytes, the mice were sacrificed after 4, 24 and 48 h; vaginae and local lymph nodes were removed, snap frozen with OCT, sectioned and examined by fluorescent microscopy and FACS. RESULTS: HIV transmission was established using virus-infected cells inoculated vaginally, as shown by FACS, HIV viral load, p24 and PCR results. Labelled cells were easily located within the vaginal tissues after 4 h. However, few or no cells could be identified after 24 or 48 h at the vaginal level, whereas labelled cells could be detected at the level of regional lymph nodes. CONCLUSIONS: Because of its simplicity and practical features compared with other animal models, the vaginal HIV-infected hu-SCID mouse model may prove useful to test the activity of compounds against cell-associated HIV and, possibly, other sexually transmitted diseases.

Cure of mice with established metastatic friend leukemia cell tumors by a combined therapy with tumor cells expressing both interferon-alpha 1 and herpes simplex thymidine kinase followed by ganciclovir.

Transduction of the murine interferon-alpha (IFN-alpha) gene into various malignant mouse tumor cells has resulted in the loss of tumorigenicity and an acquired capacity to induce long-lasting antitumor immunity following their injection into immunocompetent syngeneic mice. In the present study, we investigated the effectiveness of IFN-alpha-producing tumor cells in the therapy of mice with established mouse tumors. In DBA/2 mice bearing subcutaneous (s.c.) Friend erythroleukemia cell (FLC) tumors, we found that to achieve some antitumor response (i) it was necessary to inject high numbers of IFN-alpha-producing FLC, which occasionally lead to the formation of slowly growing tumors; and, that (ii) repeated injections of irradiated IFN-alpha-FLC did not result in any antitumor effect. The therapeutic potential of IFN-alpha-producing FLC rendered sensitive to ganciclovir (GCV), by transfer of the herpes simplex virus thymidine kinase (tk) gene, was investigated. Complete tumor rejection and cure was observed in > or = 70% of the animals after injection of high numbers (10(7)) of IFN-alpha-producing tk-expressing tumor cells followed 4 days later by repeated GCV treatments, whereas only a slight increase in survival time was obtained after administration of control tk-expressing tumor cells (not producing IFN) and GCV. Tumor rejection was associated with a dramatic destruction of tumor tissue and with the subsequent development of a potent and long-lasting antitumor immunity. No therapeutic effect was observed in immunosuppressed nude mice. These data indicate that this approach may represent an effective and safe therapeutic strategy for antitumor cytokine gene therapy.

Alterations of lipid composition in Friend leukemia cell tumors in mice treated with tumor necrosis factor-alpha.

Lipid analyses were carried out on transplantable murine Friend leukemia cell tumors, 6 h after intratumoral administration of tumor necrosis factor-alpha (TNF). The levels of the major phospholipid classes were uniformly decreased to about 70% of control values; free fatty acids were increased to about 170%; diacylglycerol was decreased to about 50% and triacylglycerol, the main lipid component, was not significantly altered. These results analysed in the light of concomitant alterations in the levels of phospholipid precursors and catabolites (determined in previous 31P NMR studies) and histological modifications demonstrated that at early stages of TNF-induced inhibition of tumor growth (a) phospholipid catabolism was significantly enhanced; (b) morphological changes were apparently correlated with alterations in the levels of phosphatidylcholine and its catabolic products.

Modulations of glycerophosphorylcholine and phosphorylcholine in Friend erythroleukemia cells upon in vitro-induced erythroid differentiation: a 31P NMR study.

A 31P NMR study has been carried out on Friend erythroleukemia cells (FLC) induced to undergo erythroid differentiation in vitro. Significant levels of glycerophosphorylcholine (GroPCho) and phosphorylcholine (P-Cho) were identified both in the untreated cells and in their PCA extracts. In FLC treated 4 days in vitro with either dimethylsulfoxide (DMSO) or hexamethylenebisacetamide (HMBA), the intracellular concentration of P-Cho was markedly increased, whereas that of GroPCho appeared to be significantly reduced. HMBA was more effective than DMSO in producing this effect. The concomitant modulations of GroPCho and P-Cho in differentiated FLC suggest the hypothesis that erythroid differentiation involves modifications of the regulatory mechanisms controlling biosynthesis and catabolism of phospholipids.

Murine interferon-alpha1 gene-transduced ESb tumor cells are rejected by host-mediated mechanisms despite resistance of the parental tumor to interferon-alpha/beta therapy.

The highly metastatic ESb tumor is totally resistant to murine interferon-alpha/beta (IFN-alpha/beta) therapy, regardless of the number of cells injected or the route of inoculation. In contrast, as we show herein, mouse IFN-alpha1-transduced ESb tumor cells were inhibited markedly when injected subcutaneously into immunocompetent mice. IFN-producing ESb tumor rejection was mediated by the immune system, because these tumor cells grew normally in immunosuppressed mice. Tumor regression was accompanied by extensive necrosis and cellular infiltrates in the tumor area. These results further support the use of IFN-alpha in cytokine gene therapy of cancer and suggest the advantage of using gene transfer rather than cytokine administration to enhance an antitumor immune response.