Daniel Benharroch

IL-1 is required for tumor invasiveness and angiogenesis

Here, we describe that microenvironmental IL-1β and, to a lesser extent, IL-1α are required for in vivo angiogenesis and invasiveness of different tumor cells. In IL-1β knockout (KO) mice, local tumor or lung metastases of B16 melanoma cells were not observed compared with WT mice. Angiogenesis was assessed by the recruitment of blood vessel networks into Matrigel plugs containing B16 melanoma cells; vascularization of the plugs was present in WT mice, but was absent in IL-1β KO mice. The addition of exogenous IL-1 into B16-containing Matrigel plugs in IL-1β KO mice partially restored the angiogenic response. Moreover, the incorporation of IL-1 receptor antagonist to B16-containing plugs in WT mice inhibited the ingrowth of blood vessel networks into Matrigel plugs. In IL-1α KO mice, local tumor development and induction of an angiogenic response in Matrigel plugs was less pronounced than in WT mice, but significantly higher than in IL-1β KO mice. These effects of host-derived IL-1α and IL-1β were not restricted to the melanoma model, but were also observed in DA/3 mammary and prostate cancer cell models. In addition to the in vivo findings, IL-1 contributed to the production of vascular endothelial cell growth factor and tumor necrosis factor in cocultures of peritoneal macrophages and tumor cells. Host-derived IL-1 seems to control tumor angiogenesis and invasiveness. Furthermore, the anti-angiogenic effects of IL-1 receptor antagonist, shown here, suggest a possible therapeutic role in cancer, in addition to its current use in rheumatoid arthritis.

Differential effects of IL-1 alpha and IL-1 beta on tumorigenicity patterns and invasiveness

In this study, we show that distinct compartmentalization patterns of the IL-1 molecules (IL-1α and IL-1β), in the milieu of tumor cells that produce them, differentially affect the malignant process. Active forms of IL-1, namely precursor IL-1α (pIL-1α), mature IL-1β (mIL-1β), and mIL-1β fused to a signal sequence (ssIL-1β), were transfected into an established fibrosarcoma cell line, and tumorigenicity and antitumor immunity were assessed. Cell lines transfected with pIL-1α, which expresses IL-1α on the membrane, fail to develop local tumors and activate antitumor effector mechanisms, such as CTLs, NK cells, and high levels of IFN-γ production. Cells transfected with secretable IL-1β (mIL-1β and ssIL-1β) were more aggressive than wild-type and mock-transfected tumor cells; ssIL-1β transfectants even exhibited metastatic tumors in the lungs of mice after i.v. inoculation (experimental metastasis). In IL-1β tumors, increased vascularity patterns were observed. No detectable antitumor effector mechanisms were observed in spleens of mice injected with IL-1β transfectants, mock-transfected or wild-type fibrosarcoma cells. Moreover, in spleens of mice injected with IL-1β transfectants, suppression of polyclonal mitogenic responses (proliferation, IFN-γ and IL-2 production) to Con A was observed, suggesting the development of general anergy. Histologically, infiltrating mononuclear cells penetrating the tumor were seen at pIL-1α tumor sites, whereas in mIL-1β and ssIL-1β tumor sites such infiltrating cells do not penetrate inside the tumor. This is, to our knowledge, the first report on differential, nonredundant, in vivo effects of IL-1α and IL-1β in malignant processes; IL-1α reduces tumorigenicity by inducing antitumor immunity, whereas IL-1β promotes invasiveness, including tumor angiogenesis, and also induces immune suppression in the host.

Contribution of IL-1 to resistance to Streptococcus pneumoniae infection

The role of IL-1 in susceptibility to Streptococcus pneumoniae infection was studied in mice deficient in genes of the IL-1 family [i.e. IL-1alpha-/-, IL-1beta-/-, IL-1alpha/beta-/- and IL-1R antagonist (IL-1Ra)-/- mice] following intra-nasal inoculation. Intra-nasal inoculation of S. pneumoniae of IL-1beta-/- and IL-1alpha/beta-/- mice displayed significantly lower survival rates and higher nasopharyngeal and lung bacterial load as compared with control, IL-1alpha-/- and IL-1Ra-/- mice. Treatment of IL-1beta-/- mice with rIL-1beta significantly improved their survival. A significant increase in blood neutrophils was found in control, IL-1alpha-/- and IL-1Ra-/- but not in IL-1beta-/- and IL-1alpha/beta-/- mice. Local infiltrates of neutrophils and relatively preserved organ architecture were observed in the lungs of IL-1alpha-/- and control mice. However, S. pneumoniae-infected IL-1beta-/-, IL-1alpha/beta-/- and IL-1Ra-/- mice demonstrated diffuse pneumonia and tissue damage. Altogether, all three isoforms contribute to protection against S. pneumoniae; our results point to differential role of IL-1alpha and IL-1beta in the pathogenesis and control of S. pneumoniae infection and suggest that IL-1beta has a major role in resistance to primary pneumococcal infection while the role of IL-1alpha is less important.

Matrix metalloproteinases 2 and 9, E-cadherin, and β-catenin expression in endometriosis, low-grade endometrial carcinoma and non-neoplastic eutopic endometrium

OBJECTIVES Endometriosis and endometrial endometrioid carcinoma are both capable of invasion and metastasis, but their biological behavior is strikingly different. Matrix metalloproteinases (MMPs) and changes in adhesion molecules have a role in the pathogenesis of various physiological and pathological processes, as well as in the development of endometriosis and endometrioid endometrial carcinoma. We hypothesized that endometriosis, being a benign process, will show different MMPs and adhesion molecules expressions, compared to endometrioid endometrial carcinoma, a disease with potential of malignant behavior. STUDY DESIGN We performed an immunohistochemical study to investigate expression of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), E-cadherin and beta-catenin in endometriosis, low-grade endometrial endometrioid carcinoma, and eutopic proliferative endometrium. Endometriotic tissues (n=15), low-grade endometrial endometrioid carcinomas (n=15), and unremarkable proliferative endometrium from women without endometriosis or carcinoma (n=10) were examined. RESULTS Endometriotic tissues showed statistically significantly stronger staining for MMP-9 and reduced beta-catenin expression when compared with proliferative endometrium. Endometrial endometrioid carcinoma showed decreased E-cadherin expression in comparison with proliferative endometrium. MMP-2 and MMP-9, and E-cadherin expressions were significantly higher and beta-catenin expression was significantly lower in endometriosis as compared to endometrioid carcinoma. CONCLUSIONS We suggest that increased MMPs and altered beta-catenin may play a role in the pathogenesis of endometriosis. Decreased E-cadherin may be important in the development of endometrial endometrioid carcinoma. The changes in MMPs, E-cadherin and beta-catenin differ in endometriosis from those in endometrioid carcinoma, an interesting finding in view of the fact that both these diseases are capable of invasion and metastasis, but have different biological behavior.

Early Peripheral Lymph Node Involvement of Human Herpesvirus 8–Associated, Body Cavity–Based Lymphoma in a Human Immunodeficiency Virus–Negative Patient

Human herpesvirus 8 (HHV-8), or Kaposi sarcoma-associated herpesvirus, is a gamma herpesvirus first detected in a specimen of Kaposi sarcoma from a human immunodeficiency virus (HIV)-positive patient. Human herpesvirus 8 is also found in an unusual clinicopathologic form of body cavity-based B-cell lymphoma, which has been named primary effusion lymphoma (PEL) and occurs primarily in HIV-positive patients. PEL is characterized by the formation of lymphomatous effusions, without obvious lymphadenopathy, tumor masses, or bone marrow involvement. Only a few cases of PEL in HIV-seronegative patients have been reported. We describe a case of an HHV-8-associated lymphoma, with ascites, pleural effusion, and axillary lymphadenopathy in an HIV-negative patient. The patient was a 68-year-old Jewish man of North African extraction, with a previous history of coronary bypass surgery and multiple blood transfusions. The pleural fluid contained large atypical lymphoid cells and was suggestive of lymphoma but could not provide a conclusive diagnosis of PEL. The lymph node contained groups of large anaplastic lymphoid cells. Polymerase chain reaction for HHV-8 performed on the lymph node specimen was positive, establishing the diagnosis of PEL. Polymerase chain reaction for Epstein-Barr virus was negative. Results of a gallium scan were normal. The patient did not respond to combination chemotherapy with cyclophosphamide, doxorubicin, vincristine sulfate, and prednisone and progressively developed, massive intra-abdominal solid tumor formation. To our knowledge, this is the first report of a case of PEL that demonstrates peripheral lymph node involvement at diagnosis and the first report of PEL in an Israeli patient.

Osteoblastic osteitis of the maxillary sinus

Osteoblastic osteitis is a rare kind of bone infection typified by a proliferative reaction of the periosteum and by exuberant bone formation. In the maxillary sinus, it occurs as a consequence of chronic or recurrent sinusitis. It usually manifests with a vague facial discomfort, followed by complications in the deep facial spaces or fossae. The diagnosis is a radiological one. Eradication of this bone infection necessitates removal of the precipitating condition as well as the long‐term administration of appropriate antibiotics. In the case of a deep facial fossae abscess, drainage is mandatory.

Differential activation of the immune system by virulent Streptococcus pneumoniae strains determines recovery or death of the host.

Streptococcus pneumoniae infection may result in asymptomatic carriage, mucosal or invasive disease. We hypothesize that self‐limiting or fatal disease outcome follows infection with S. pneumoniae differential activation of the host immune response. BALB/c and C57BL/6 mice were inoculated intranasally with S. pneumoniae serotype 3 strain WU2 and serotype 14 strain DW14 and mortality, bacterial load, pathological changes in the lungs and cytokines mRNA levels in the spleen were analysed. No differences between the C57BL/6 and the BALB/c inbred mice were observed except for the severity of their lung pathology and IL‐4 expression. Infection of the two mouse strains with S. pneumoniae WU2 resulted in sepsis and death that occurred within 4 days post‐inoculation. This death was preceded, in both mouse strains, in an increase over time of the lung bacterial load and bacteraemia. The lung pathology was characterized by diffuse pneumonia with marked congestion of the lungs. Analysis of mRNA expression of cytokines in the spleen revealed no alterations in tumour necrosis factor (TNF)‐α, transforming growth factor (TGF)‐β, interleukin (IL)‐12 and interferon (IFN)‐γ and induction of IL‐10 and IL‐4. The two strains of mice survived infection with S. pneumoniae DW14. This was accompanied by a reduction over time of lung bacterial load and bacteraemia. The lung pathology was characterized by focal lymphocyte infiltration and preserved architecture of the organ. Analysis of mRNA expression of cytokines in the spleen revealed a significant decrease in the levels of TNF‐α, TGF‐β, IL‐12 and IFN‐γ mRNA expression, which usually precedes cytokine protein expression. Interestingly, a significant increase in the levels of IL‐4 mRNA expression was found in BALB/c mice only.  This study suggests that differential activation or evasion of cytokine expression by S. pneumoniae virulent strains determines disease outcome regardless of the hosts immunogenetic background.

The neuropathology of cerebrotendinous xanthomatosis revisited: a case report and review of the literature

Cerebrotendinous xanthomatosis (CTX), a rare autosomal-recessive lipid storage disease, has been well characterized clinically and biochemically, and recently also from the molecular biological aspect. However, only a very few publications deal with its neuropathology, and views on its pathogenesis vary. Based on a recently examined, case, we propose that central-peripheral distal axonopathy is the major pathogenetic mechanism of nervous system injury in CTX. The latter is characterized by white matter pathology, typically in form of long tract involvement with the more distal parts of the tract more severely affected. Most severely affected are the cerebellar white matter, the optic pathways and the long tracts of the brain stem and spinal cord, particularly the pyramidal tracts, although there is hardly a CNS region which does not display some form of pathology. Lesions are characterized by loss of myelinated fibers and accumulation of lipid products in form of foamy macrophages, clear oil-red-O-positive spaces and crystalline clefts, accompanied by gliosis, occasional axonal spheroids, and in the cerebellum — the most severely affected structure — also by multinucleated foreign body giant cells. Demyelination is not seen, and ultrastructurally myelin sheaths are normally structured. Signs of axonal degeneration are also present in the spinal roots. We hypothesize that the basic enzymatic defect in CTX leads to accumulation of metabolites in the brain which may be neurotoxic and may impair the metabolic apparatus of neurons with resultant axonopathy and subsequent nonspecific lipid deposition in the injured tracts.

Nuphar lutea thioalkaloids inhibit the nuclear factor κappaB pathway, potentiate apoptosis and are synergistic with cisplatin and etoposide

We screened thirty-four methanolic plant extracts for inhibition of the constitutive nuclear factor κB (NF-κB) activity by a NF-κB-luciferase reporter gene assay. Strong inhibition of NF-κB activity was found in extracts of leaf and rhizome from Nuphar lutea L. SM. (Nuphar). The inhibitory action was narrowed down to a mixture of thionupharidines and/or thionuphlutidines that were identified in chromatography fractions by one- and two-dimensional NMR analysis. Dimeric sesquiterpene thioalkaloids were identified as the major components of the mixture. The Nuphar alkaloids mixture (NUP) showed a dose dependent inhibition of NF-κB activity in a luciferase reporter gene assay as well as reduction of nuclear NF-κB subunits expression as tested by western blots and immunohistochemistry. Decreased DNA binding was demonstrated in electro mobility shift assays. NUP inhibited both inducible and constitutive NF-κB activation and affected the canonical and alternative pathways. Suppression of NF-κB was not cell type specific. Induction of apoptosis by the alkaloid mixture was demonstrated by time-dependent and dose-dependent cleavage of procaspase-9 and PARP. Synergistic cytotoxicity of the active mixture with cisplatin and etoposide was demonstrated. Overall, our results show that NUP inhibits the NF-κB pathway and acts as a sensitizer to conventional chemotherapy, enabling the search for its specific target and application against cancer and inflammation.

PKCη Expression Contributes to the Resistance of Hodgkin's Lymphoma Cell Lines to Apoptosis

The Hodgkin-Reed-Sternberg (HRS) malignant cells in Hodgkins lymphoma (HL) originate from germinal center B lymphocytes that did not undergo apoptosis. Protein Kinase C (PKC), a family of serine/threonine kinases, plays a crucial role in signal transduction modulating cell growth, differentiation and apoptosis. Here, we report the expression of PKC isoforms in two HL-derived cell lines, L428 and KMH2 and their correlation with drug resistance to CPT and doxorubicin. Among the PKC isoforms examined, only PKCη and PKCβΙΙ were preferentially expressed in the drug resistant L428 cells. We have shown correlation between the response to apoptosis of L428 and KMH2 cells and PKCη expression in these cell lines. In order to directly demonstrate a role for PKCη in apoptosis, its expression was knocked-down by siRNA in the resistant L428 cells. Down-regulation of PKCη rendered L428 cells more sensitive to doxorubicin and CPT. Furthermore, PKCη knocked-down cells showed increased PARP-1 cleavage, cytochrome c release and caspase 7 activation. It appears that PKCη functions as an anti-apoptotic protein in HL-derived cell lines, and as we show here that it is also expressed in HRS of HL biopsies, it may have therapeutic relevance in HL. Thus, PKCη could provide a new target aimed to reduce resistance to anti-cancer treatments of HL and other cancer patients.