Primary thrombocythemia in a female carrier of IgA deficiency]

We describe a case of essential thrombocythemia observed in a 67-year-old woman with severe IgA-deficiency. The the best of our knowledge, this is the first report concerning the onset of a chronic myeloproliferative disease (CMPD) in a patient affected with primary immunodeficiency, in particular IgA-defect. The association may be merely coincidental; otherwise hemopoietic growth factors acting on myeloid progenitor cells could play a role in this relationship. It has recently been shown that serum levels of many cytokines are elevated in patients with CMPD and probably contribute to enhance proliferation of the malignant clones; on the other hand interleukin-6 seems to account for reactive thrombocytosis, and significant amounts of circulating interleukin-4 and interleukin-6 have been detected in IgA-deficient patients. Overproduction of the two cytokines may depend on recurrent infections, but it could also represent a primary abnormality, with a putative role in the pathogenesis of the immune defect. These findings suggest that high levels of growth factors could induce myeloid hyperproliferation and so expose stem cells to genetic mutations responsible for malignant transformation.     

Immunological and side effects of low sc recombinant interleukin-2 dose in addition to conventional treatment in relapsed breast and colorectal cancer patients

Thirteen relapsed cancer patients, four of them operated for colorectal and the nine remaining for breast cancer, were cyclically given low subcutaneous (sc) recombinant interleukin-2 (rIL-2) doses in addition to chemo- or hormone therapy. Cycle intervals were 2 or 6 weeks in length, and the number of cycles ranged from one to 14 and from one to six respectively. Tolerance assessed by clinical and laboratory data, eosinophils, lymphocytes (total number), T subpopulations, B lymphocytes and NK cells were the evaluated parameters. One (7.6%) of the 13 studied patients interrupted the first low dose sc rIL-2 cycle due to a hypersensitive reaction. This case showed relapse from breast cancer. During further cycles, three patients (25%), one operated on for colorectal and two others for breast cancer of the 12 remaining cases who completed all rIL-2 cycles showed an increase in glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), gamma-gt, and creatininemia without any clinical symptoms. A slight influenza-like syndrome and 10-20 mmHg decrease in blood pressure sporadically occurred in all patients under rIL-2 therapy. In both cancer types, a significant (P < 0.05 - P < 0.001) increase in lymphocytes, eosinophils. T4 and T3 subpopulations but not in T8 subpopulations and NK cells occurred at the end of the rIL-2 cycles. In 11 of the 13 patients responsive to conventional therapy, a highly significant increase (P < 0.001) in all parameters apart from B lymphocytes and T4/T8 ratio was observed, while in three cases which were no longer responsive and in another case which had never been responsive to conventional therapy, a slightly significant increase in eosinophils only occurred (P < 0.05). Three colorectal cancer patients showed a partial response and the last a complete response to conventional therapy. In these four patients, time to progression during rIL-2 cycles ranged from 2.5-5 months and the duration of response ranged from 8-19 months. In seven of the eight breast cancer patients who completed all rIL-2 cycles, the response ranged from 3-51+ months and in the last case, which was not responsive to conventional therapy, the disease progressed in spite of the addition of rIL-2. These data suggest that: a) rIL-2 is likely to constitute a well-tolerated and suitable home therapy even when cyclically given for a prolonged period; b) following rIL-2 administration, eosinophils and lymphocytes increase in addition to the T subpopulations.     

Effect of endogenous proteins on growth and antibody productivity in hybridoma batch cultures

It has been shown that some B-cell hybridomas secrete autocrine factors in vitro which can influence cell metabolic processes. Rather than screen specifically for suspected cytokines, that may or may not affect our cell line, we have examined the lumped effects of intracellular and secreted factors on cell proliferation and monoclonal productivity in hybridoma batch cultures. Firstly, supplements of total soluble intracellular proteins combined with other intracellular metabolites were found to both decrease the specific growth rate and increase the antibody production rate at higher concentrations in batch culture. This is an important consideration in high cell density cultures, such as perfusion systems, where a reduction of growth by the presence of intracellular factors may be compensated by an increase in MAb production. In addition, flow cytometry data revealed that the average cell cycle G1 phase fraction was unaffected by the variation in the maximum specific growth rates during the exponential growth phase, caused by the addition of intracellular factors; this suggests that higher MAb productivity at lower growth rates are not a result of cell arrest in the G1 phase. Secondly, secreted extracellular proteins larger than 10,000 Daltons, which were concentrated from spent culture supernatant, were shown to have no significant effect on growth and specific MAb productivity when supplemented to batch culture at levels twice that encountered late in normal batch culture. This indicates that endogenous secreted cytokines, if at all present, do not play a major autocrine role for this cell line.     

Identification of an IL-7-associated pre-pro-B cell growth-stimulating factor (PPBSF). I. Production of the non-IL-7 component by bone marrow stromal cells from IL-7 gene-deleted mice

Mouse bone marrow (BM) stromal cell conditioned medium (CM) from our long-term lymphoid culture system selectively induces the in vitro proliferation and presumptive differentiation of pre-pro-B cells (B220+, HSA-, TdT- or TdT+, c[mu-]) from adult rat, mouse, and human BM. However, the responsible growth factor(s) has not yet been identified. Inasmuch as IL-7 is one of the cytokines most closely associated with early B-lineage development, we utilized BM adherent cells and stromal cell lines from IL-7 gene-deleted (-/-) mice in combination with rIL-7 and anti-IL-7 mAb to investigate its possible regulatory role in our culture system. The results show that, although rIL-7 and IL-7 (-/-) CM each can maintain the viability of freshly harvested pre-pro-B cells in vitro, neither induces them to proliferate and/or differentiate, even in the presence of recombinant stem cell factor (rSCF) and/or recombinant insulin-like growth factor (rIGF). The results also show that anti-IL-7 mAb fails to neutralize the pre-pro-B cell growth-stimulating activity in IL-7 (+/+) CM. Yet rIL-7 enables IL-7 (-/-) CM to induce proliferation of pre-pro-B cells, and to "prime" them to respond directly to monomeric IL-7. Furthermore, anti-IL-7 mAb adsorbs the pre-pro-B cell growth-stimulating activity from both IL-7 (+/+) CM and rIL-7-supplemented IL-7 (-/-) CM; but rIL-7 does not restore this activity. Lastly, both pre-pro-B cell growth-stimulatory activity and IL-7 are quantitatively recovered by ultrafiltration in the 50 to 100 kDa, rather than the 10 to 50 kDa, apparent molecular mass fraction. These results suggest that the pre-pro-B cell growth-stimulating activity in our culture system is the property of a self-associating complex of IL-7 and a second BM stromal cell-derived cofactor.     

Cytokines in acute myeloid leukemia]

The cytokines belong to the group of main factors regulating of leukaemia cell proliferation. Most of information comprised in the literature concern the behaviour of single cytokines in the cell culture in vitro. In the organism of leukaemia patient many different cytokines, adhesion molecules, growth factors and other substances probably act in the same time. Therefore the aim of study was the determination of plasma concentrations of interleukin 1B, 3, 4, 6, 8, G-CSF and P-selection in 26 patients with acute myeloblastic leukaemia-aml, among them 14 patient in the course of exacerbation-aml-e and 12 being in the remission-aml-r, classified as type M1 and M2 according to the FAB classification. Control group consisted of 15 healthy volunteers. The cytokine measurements were performed by means of immunoradiometric, immunoenzymatic and radioimmunologic kits. In the patients with aml-e significant increase of plasma concentrations of IL-1B, IL-3, IL-6, G-CSF, P-selectin and essential decrease of IL-4 was found. Significant decrease of IL-4 and P-selecting concentrations in the patients with aml-r and lack of changes in IL-8 concentrations in the both groups of patients was demonstrated. The observed differences of concentrations may additionally confirm the role of studied cytokines and P-selectin in the regulation of leukaemia cell proliferation.     

IL-6 rescues resting mouse T cells from apoptosis

It has previously been demonstrated that mature mouse T cells live for many weeks in vivo. In contrast, explanted lymph node or splenic T cells undergo spontaneous death within days, suggesting that survival factors supplied in vivo are not present in normal tissue culture medium. We discovered that IL-6 can rescue resting T cells from apoptosis in vitro. We show that recombinant mouse IL-6 as well as IL-6 in endothelial cell supernatants are sufficient to rescue T cells from death in the absence of additional cytokines. We show that CD4+ T cells express Bcl-2 immediately following isolation from the mouse, but after 24 h in culture Bcl-2 is undetectable. If during this time period the T cells are incubated with rIL-6, Bcl-2 expression is not down-regulated. It is, therefore, possible that IL-6 rescue from death is mediated by maintenance or induction of Bcl-2 expression. Addition of rIL-6 does not by itself induce blastogenesis or proliferation, and therefore, this cytokine appears to be a true survival factor rather than a mitogenic factor for resting T cells. Together, these results support a potential role for IL-6 as one of the factors important for prolonging resting T cell survival in vivo.     

Congenital cholesteatoma of temporal bone

Cytomegalovirus infection causing symptomatic enteritis is most usually associated with immunosuppressed transplant patients or patients positive for human immunodeficiency virus. Most reports studying this illness are small and do not clearly define the risk factors or mortality rates. METHODS: The present study retrospectively reviewed the charts of 67 patients with biopsy-proven cytomegalovirus enteritis (esophageal, gastric, small bowel, and colonic) to define and to investigate factors that influence survival. Patients were classified into four groups based on underlying medical condition: 1) patients positive for human immunodeficiency virus; 2) transplant patients receiving immunosuppressive medications; 3) immunosuppressed nontransplant patients; and 4) otherwise healthy individuals. Mortality rates based on underlying medical condition, location of intestinal cytomegalovirus infection, cytomegalovirus therapy, age, and average days to institution of treatment were defined and statistically assessed. RESULTS: Mortality was significantly greater in the normal patient group (80 percent) than in the transplant (21 percent), other immunosuppressed (44 percent), or human immunodeficiency virus-positive (75 percent) groups (P = 0.0006, Cochran-Mantel-Haenszel statistics). There was no difference in mortality based on intestinal location of disease or treatment modality (surgery, medical therapy, or both). Cohorts of patients older than 65 years had a statistically higher mortality rate vs. those younger than 65 years old (68 vs. 38 percent; P = 0.05, Cochran-Mantel-Haenszel statistics). Statistically increased mortality was also associated with increased time from hospital admission to institution of cytomegalovirus treatment, whether therapy was medication alone or medication and surgery (P < 0.05, exact Wilcoxon's test). CONCLUSIONS: 1) Lethal cytomegalovirus enteritis can arise in patient populations not typically identified as being at risk for this disorder, including normal individuals. 2) Mortality in cytomegalovirus enteritis is adversely associated with age older than 65 years and increased time to institution of therapy but is not affected by anatomic site of infection or particular form of treatment. Paradoxically, in this study, normal patients had the highest mortality, which we attribute to a low index of suspicion and relatively late institution of therapy.     

Human T cells require IL-2 but not G1/S transition to acquire susceptibility to Fas-mediated apoptosis

The interaction between Fas ligand and Fas, both expressed on activated T cells, is the major pathway in the regulation of activation-induced cell death. However, activated T cells that express membrane Fas are initially resistant to anti-Fas-induced apoptosis and become susceptible only after proliferation in vitro. Since IL-2 is known to regulate activation-induced cell death, we studied the effect of IL-2 on anti-Fas-mediated apoptosis. Interference with the IL-2 pathway was achieved by 1) inhibition of cytokine synthesis using cyclosporin A or FK506, 2) neutralization of IL-2 by anti-IL-2 Ab, 3) inhibition of binding to IL-2R by CD25 mAb, and 4) blocking of IL-2R signaling by rapamycin. We show that Fas expression is independent of the IL-2 pathway, whereas Fas-mediated apoptosis does not develop in the presence of inhibitors of IL-2 production or signaling. While the addition of rIL-2 reversed the inhibitory effect of cyclosporin A and FK506, the addition of rIL-4, rIL-7, or rIFN-gamma did not, although these cytokines induced progression into the S phase of the cell cycle. Aphidicolin-treated activated T cells that do not progress into the S phase were susceptible to Fas-mediated apoptosis. Therefore, Fas-mediated apoptosis is controlled by signals generated by IL-2 in agreement with the reported alteration of apoptosis in mice deficient in IL-2 or IL-2R.     

Regulation of 12-lipoxygenase by cytokines in vascular smooth muscle cells

Increasing evidence suggests that cytokines such as interleukin-1beta (IL-1), IL-4, and IL-8 may play an important role in the chronic inflammation and cellular growth observed in cardiovascular diseases. The lipoxygenase (LO) pathway of arachidonate metabolism has also been related to the pathology of hypertension and atherosclerosis. LO products have chemotactic, hypertrophic, and mitogenic effects in vascular cells, and the LO enzyme has been implicated in the oxidation of LDL. Furthermore, earlier studies have shown that vascular smooth muscle cell (VSMC) growth factors such as angiotensin II and platelet-derived growth factor can increase LO activity and expression in VSMCs. In the present study, we have examined whether vasoactive and inflammatory cytokines such as IL-1, IL-4, and IL-8 can modulate 12-LO activity and expression in porcine VSMCs and also whether they have growth-promoting effects in these cells. Treatment of porcine VSMCs with these cytokines led to significant increases in the levels of a cell-associated 12-LO product, 12-hydroxyeicosatetraenoic acid, as well as intracellular 12-LO enzyme activity. Furthermore, each of these cytokines led to a dose-dependent increase in 12-LO mRNA expression (333-base pair PCR product) as well as 12-LO protein expression (72 kD). In addition, all three interleukins could induce significant increases in VSMC DNA synthesis as well as proliferation. These results suggest that these cytokines have mitogenic effects in VSMCs and are also potent positive regulators of the 12-LO pathway. Thus, enhanced 12-LO activity and expression may be a key mechanism for cytokine-induced VSMC migration and proliferation.     

An anti-IL-2 antibody increases serum half-life and improves anti-tumor efficacy of human recombinant interleukin-2

We examined the ability of anti-human recombinant interleukin-2 (hu rIL-2) monoclonal antibody DMS-1.10 to increase serum half-life of hu rIL-2, and the effect of this complex on inhibition of tumor progression in a B16-F10 murine melanoma model. In C57B1/6 mice, intravenous (i.v.) injection of DMS-1.10 premixed with 1 x 10(4) units (U) of hu rIL-2 at a 1:1 molar ratio extended serum half-life greater than 10-fold (222 min) when compared to the same dose of hu rIL-2 alone (20 min). In a murine tumor model, multiple intraperitoneal (i.p.) injections of non-neutralizing DMS-1.10 premixed with hu rIL-2 at a 5:1 molar ratio reduced the growth rate of subcutaneous (s.c.) B16-F10 tumor in C57B1/6 mice by 64% when compared to PBS and irrelevant antibody treated controls. Although similar treatment with hu rIL-2 alone reduced tumor growth rate by 46%, it was significantly less effective than the premixed treatment. Results from a flow cytometry assay confirm B16-F10 does not have IL-2 receptors, precluding direct inhibition of tumor growth by hu rIL-2 treatments. We propose that therapeutic efficacy of hu rIL-2 is improved by prolonging the in vivo half-life with an anti-IL-2 antibody, thus augmenting hu rIL-2 bioactivity and enhancing the hosts immune response against tumor.     

Therapy of primary central nervous system lymphoma with pre-irradiation methotrexate, cyclophosphamide, doxorubicin, vincristine, and dexamethasone (MCHOD)

Recently, Khayat et al. reported that high-dose recombinant interleukin-2 (rIL-2) i.v. may induce tumour regressions in metastatic melanoma patients through an association with cisplatin (CDDP) and alpha-interferon (alpha-IFN). Treatment-related toxicities are, however, important. Previous studies have demonstrated that rIL-2 toxicity may be reduced through a subcutaneous injection. In order to evaluate the effectiveness of low subcutaneous rIL-2 doses in a chemoimmuno-hormonotherapeutic combination, 36 metastatic melanoma patients were treated with CDDP, rIL-2, alpha-IFN and tamoxifen (TAM). The overall response rate was 47.2%: five patients had complete response (14%), 12 partial response (33%) and 13 stable disease (36%). Median response duration was 6.4 months (range: 2-29+). Median overall survival was 10 months (range: 3-36+). The CDDP/rIL-2/alpha-IFN/TAM regimen was effective both on soft tissue and visceral metastases. Toxicity was low and patient management did not require an intensive care unit. A statistically significant increase in both percentage and absolute values of lymphocytes, eosinophils, CD3+/CD4+, CD25+, CD16/56+ and HLA-DR+ cells was found in all patients after two treatment courses. This study shows that lower doses of subcutaneous rIL-2, as well as CDDP and alpha-IFN, associated with TAM, may have similar anticancer efficacy with respect to Khayat's schedule but lower toxicity.     

IL-15 augments CD8+ T cell-mediated immunity against Toxoplasma gondii infection in mice

Cytokines of the Th1 profile are important mediators of protective host immunity against Toxoplasma gondii infection in mice. In this study we describe the effect of the recently identified cytokine, IL-15, on prevention of murine infection with T. gondii. Administration of exogenous rIL-15 with soluble Toxoplasma lysate Ag (TLA) provides complete protection against a lethal parasite challenge, whereas treatment with either rIL-15 or TLA alone is not protective. Following immunization with TLA/rIL-15, there is a significant proliferation of splenocytes expressing the CD8+ phenotype in response to TLA. A significant rise in the level of serum IFN gamma was observed in vaccinated mice. Adoptive transfer of CD8+ T cells, but not CD4+ T cells, from TLA/rIL-15-vaccinated mice protects naive mice from a lethal parasite challenge. These CD8+ T cells exhibit enhanced CTL activity against target macrophages infected with T. gondii. Mice that have been immunized are protected against lethal parasite challenge for at least 1 mo postvaccination. These observations demonstrate that TLA when administered with exogenous rIL-15 generates toxoplasmacidal Ag-specific CD8+ T cells. These T cells proliferate upon exposure to parasite Ag, exhibit long term memory CTL against infected target cells, and may be involved in host immune memory to this parasite.     

Efficacy of antitat gene therapy in the presence of high multiplicity infection and inflammatory cytokines

Because human immunodeficiency virus type 1 (HIV-1) infection is characterized by a large number of viral replication cycles and rapid cell turnover in vivo, successful gene therapy requires an approach effective under these conditions. The antitat gene has been proposed for gene therapy because it effectively blocks Tat function and the replication of HIV-1. However, neither antitat nor any other antiviral gene has been shown to inhibit HIV in the presence of high viral load and inflammatory cytokines, a condition closer to the in vivo situation. We show that cells transduced with antitat retrovirus vector are resistant to high multiplicity of HIV infection. In the presence of inflammatory cytokines, including interleukin-1 and tumor necrosis factor, both known to activate viral gene expression independently of Tat, antitat suppressed virus replication. HIV-1 inhibition was observed when cell were treated with a mixture of inflammatory cytokines able to induce acquired immunodeficiency syndrome (AIDS) Kaposi's sarcoma cell growth. These molecules have been shown to be increased in HIV-1-infected individuals, and it is suggested they play a role in the pathogenesis of AIDS. Our results suggest that antitat is effective under conditions present in vivo and therefore a primary candidate for HIV-1 gene therapy.     

Epidemiology and pathogenesis of AIDS-related lymphomas

Among patients with congenital and acquired immunodeficiencies, non-Hodgkin's lymphoma (NHLs ) are the most common tumors of the immune system. In the setting of human immunodeficiency virus (HIV) infection, as many as 10% to 20% of people ultimately developed NHLs. These tumors are clinically aggressive, frequently involve extranodal sites, and often exhibit unique features that distinguish them from NHL arising in individuals with other forms of immunosuppression. Important in the development of HIV-associated NHL are cytokines and other factors that induce B-cell proliferation and increase the likelihood of mutations of c-myc, bcl-6, and other tumor-suppressor genes with carcinogenic potential. Specific forms of HIV-associated NHL are linked to expression of Epstein-Barr virus (EBV)-latent proteins; the newly described DNA virus, Karposi's sarcoma-associated herpesvirus/human herpesvirus-8 (KSHV/HHV-8); and perhaps HIV. Elucidation of the factors that contribute to the high incidence of NHL among patients infected with HIV provides insights into important elements of lymphomagenesis.     

Neurotrophic factors increase axonal growth after spinal cord injury and transplantation in the adult rat

The capacity of CNS neurons for axonal regrowth after injury decreases as the age of the animal at time of injury increases. After spinal cord lesions at birth, there is extensive regenerative growth into and beyond a transplant of fetal spinal cord tissue placed at the injury site. After injury in the adult, however, although host corticospinal and brainstem-spinal axons project into the transplant, their distribution is restricted to within 200 micron of the host/transplant border. The aim of this study was to determine if the administration of neurotrophic factors could increase the capacity of mature CNS neurons for regrowth after injury. Spinal cord hemisection lesions were made at cervical or thoracic levels in adult rats. Transplants of E14 fetal spinal cord tissue were placed into the lesion site. The following neurotrophic factors were administered at the site of injury and transplantation: brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), ciliary-derived neurotrophic factor (CNTF), or vehicle alone. After 1-2 months survival, neuroanatomical tracing and immunocytochemical methods were used to examine the growth of host axons within the transplants. The neurotrophin administration led to increases in the extent of serotonergic, noradrenergic, and corticospinal axonal ingrowth within the transplants. The influence of the administration of the neurotrophins on the growth of injured CNS axons was not a generalized effect of growth factors per se, since the administration of CNTF had no effect on the growth of any of the descending CNS axons tested. These results indicate that in addition to influencing the survival of developing CNS and PNS neurons, neurotrophic factors are able to exert a neurotropic influence on injured mature CNS neurons by increasing their axonal growth within a transplant.