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There are conflicting reports concerning the prevalence of Mycoplasma fermentans in HIV-positive patients and its association with AIDS. Serum antibodies to M. fermentans were measured by a modified immunoblotting technique in 48 HIV-positive heterosexual patients and in 30 HIV-negative heterosexual controls. Antibodies to M. fermentans were detected in 19 (40%) of HIV-positive patients and in three (10%) of the HIV-negative controls ( P =0.01). The prevalence of antibodies to Mycoplasma hominis and to Ureaplasma urealyticum was similar in both groups. In the HIV-positive group, 16/19 (84%) M. fermentans -positive patients developed AIDS, compared to eight of 29 (28%) M. fermentans -negative patients ( P =0.0004). The HIV-positive patients with antibodies to M. fermentans had a lower CD4+ cell count and a higher prevalence of antibodies to the other mycoplasma tested ( P =0.007 and P =0.03, respectively), as compared to the patients without antibodies to M. fermentans . These findings may suggest that the presence of antibodies to M. fermentans indicate an opportunistic infection. Of the 19 M. fermentans -positive patients, 11 were positive on the first examination, and eight became positive during the follow-up period. Seven out of these eight patients developed antibodies to M. fermentans before the development of AIDS. Therefore, the possibility exists that M. fermentans might influence the development of AIDS.

The data collected for the second edition of the Directory of Medical Research in Israel and the Israel Biomedical Database have yielded very relevant information concerning the distribution of investigators, publication activities and funding sources. The aggregate data confirm the findings of the first edition published in 1996 [2]. Those facts endorse the highly concentrated and extensive nature of medical research in the Jerusalem area, which is conducted at the Hebrew University and its affiliated hospitals. In contrast, Tel Aviv University, whose basic research staff is about two-thirds the size of the Hebrew University staff, has a more diffuse relationship with its clinical staff who are located at more than half a dozen hospitals. Ben-Gurion University in Beer Sheva and the Technion in Haifa are smaller in size, but have closer geographic contact between their clinical and basic research staff. Nonetheless, all the medical schools and affiliated hospitals have good publication and funding records. It is important to note that while some aspects of the performance at basic research institutions seem to be somewhat better than at hospitals, the records are actually quite similar despite the greater burden of clinical services at the hospitals as compared to teaching responsibilities in the basic sciences. The survey also indicates the substantial number of young investigators in the latest survey who did not appear in the first survey. While this is certainly encouraging, it is also disturbing that the funding sources are apparently decreasing at a time when young investigators are attempting to become established and the increasing burden of health care costs precludes financial assistance from hospital sources. The intensity and undoubtedly the quality of medical research in Israel remains at a level consistent with many of the more advanced western countries. This conclusion is somewhat mitigated by the fact that there is a decrease in available funding and a measurable decrease in scholarly activity at a time when a new, younger generation of investigators is just beginning to become productive. In closing, we wish to stress that the collection of data for the Biomedical Database is a continuing project and we encourage all medical researches who may not have contributed relevant information to write to the Office of the Chief Scientist or contact the office by email.

Measles virus (MV) is among the infectious agents displaying a propensity for establishing persistent infections of the CNS. It is assumed that continuous presence of MV defective particles or viral genome in persistently infected cells may influence host cellular processes and perturb biochemical signal transduction pathways operating in linkage to various cell surface receptors. PKC expression in a MV persistently infected neuroblastoma cell line (NS20Y/MS) was investigated. The relative levels of PKC isoenzymes were determined by Western blot analysis. We found that protein levels of PKCalpha, epsilon and zeta, but not PKCdelta, were increased in NS20Y/MS cells. PKCbeta, gamma and eta were undetectable. Treatment of NS20Y/MS cells with anti-MV Abs, which downregulated MV protein synthesis, also reduced PKCalpha expression to the basal level observed in the uninfected NS20Y cells. Our results suggest that a persistent MV infection has specific effects on the expression of certain PKC isoenzymes. We postulate that the MV-associated neurologic changes may reflect virus induced changes in biochemical signaling pathways and that these effects are likely to be regulated by the host's anti-viral humoral immune response.

Application of neutralizing anti-hemagglutinin antibodies to mouse neuroblastoma cells (NS20Y/MS) persistently infected with measles virus (MV) leads to a significant reduction of viral structural proteins within 6 days. While the transcriptional gradient for MV-specific mRNAs remained unaffected upon antibody treatment, the total amount of MV-specific transcripts dropped by 80% after 24 h. The expression of genomic RNA was affected similarly, with slightly slower time kinetics. Both transcription and expression of the viral structural proteins could be completely reactivated when viral antibodies were removed from the tissue culture. The same findings could be obtained in rat glioma cells persistently infected with subacute sclerosing panencephalitis virus (C6/SSPE) but not in cells of nonneural origin. The data indicate that antibody-induced antigenic modulation affects the early stages of viral transcription within a few hours after the addition of antibodies and leads to an almost complete repression of viral gene expression in cells of neural origin.

In previous studies, we have demonstrated that the highly metastatic IE-7 cell clone, derived from the T-10 fibrosarcoma, expressed both the H-2Dk and H-2Db genes, whereas a nonmetastatic IC-9 clone, derived from the same tumor, expressed only H-2Db, suggesting that the H-2Dk product might be involved in the metastatic phenotype. To substantiate this notion, IC-9 cells were transfected with an H-2Dk-expressing vector. Although all of the 4 randomly selected transfectant subclones elicited high H-2Dk expression, only one was as metastatic as IE-7 cells. This metastatic transfectant resembled IE-7 cells also in its inability to evoke CTL response in syngeneic mice, whereas the other transfectants were quite competent in this respect. It thus appears that the H-2Dk product may contribute to the metastatic phenotype provided that it is immunogenically abnormal. In addition, the present study provides evidence to suggest that lack of production of the tissue inhibitor of metalloproteinases TIMP-1/TIMP-2 is another important determinant in the metastatic phenotype of these cells.

We have used the murine 3-methylcholanthrene induced T10 fibrosarcoma tumor cell system originating in (C3II/en x C57BL/6)F1 mice (H-2b x H-2k) to elucidate the possible correlation between metastatic potential, expression of individual H-2 antigens and susceptibility to NK cells. Transfection of the non metastatic and NK sensitive IC9 cells (Db+, Kk, Kb, Kk-) with the H-2Dk gene, altered the metastatic phenotype of the parental cells, yet had no effect on the susceptibility of these tumor cells to lysis by NK and did not elicit a specific CTL response in syngeneic hosts. Variants of the metastatic and NK resistant IE7 clone (Db+, Kk-, Kb-, Kk-), lacking H-2Dk, were selected by treatment with monoclonal anti H-2Dk antibodies and complement. These variants were sensitive to NK and poorly or non metastatic. Transfection of Dk negative variants with the H-2Dk gene, resulted in the isolation of several clones which expressed a wide range of metastatic phenotypes but maintained sensitivity to NK. In addition, by cloning the cDNA of the H-2Dk gene of the metastatic T10-IE7 variant cells and analyzing its nucleotide sequence, we found four single nucleotide changes. Two of them are not expected to alter the encoded amino acids, whereas the others should result in two amino acid substitutions in the alpha-2 domain of the class I H-2Kd protein product. These changes might account, at least partially, for the failure of the transfection of H-2Dk to restore resistance to NK.(ABSTRACT TRUNCATED AT 250 WORDS)

MS is among the infectious agents known to persistently infect cells of the CNS. Clones NS20/Y and NS20/MS persistently infected with MS, both originating from the C1300 mouse neuroblastoma, were used. Multiple effects of the MS infection on the neuronal cell communication, expression of protooncogenes tumorigenicity and on the presence of immunoregulatory molecules were studied. Our results demonstrate that the level of the MHC class I and II antigens and beta-2 microglobulin was elevated in the MS infected cells. Furthermore, MS infection results in the significant increase of protein kinase C (PKC) activity concomitantly with the elevation of PKC-I specific m-RNA. The MS infection was found to affect also the expression of the protooncogenes known to associate with the PKC signaling system. Thus, the level of c-fos mRNA was elevated in the MS infected cells, while there were almost no changes in the c-myc gene expression. Ki-ras and Ha-ras appeared to be regulated differently by MS infection. The level of Ki-ras mRNA was unchanged, but the expression of the Ha-ras gene was markedly depressed, correlating well with the low tumorigenicity of the MS infected neuroblastoma cells in nude mice. Our results suggest that viral infection may be beneficial in certain cases of depressing oncogenic genes which may contribute to the development and maintenance of the malignant phenotype.

Many experiments have shown convincingly that natural killer (NK) cell activity against viral infections is an important early defence mechanism in mice. Since the NK response occurs soon after infection, often long before clinical signs of disease become manifest, it has been difficult to design studies to monitor accurately NK cell kinetics following infection, without actually administering pathogens to volunteers. There is therefore little data pertaining to the role of NK cells in humans. Nevertheless, a number of studies have shown elevated NK activity in response to herpes simplex and influenza virus infections in humans. Our study was designed to show that NK activity could be provoked in humans by exposure to viral particles without actual live viral infection. The development of NK cell response in the peripheral blood of volunteers shortly after vaccination with killed influenza trivalent vaccine was studied. The results demonstrate that killed virus vaccine induces and augments NK cell activity for relatively long periods. Such data may prove valuable for designing possible modes of augmenting NK activity as a therapeutic tool.

Silica specifically kills macrophages in vitro, and in vivo has been used as a method of determining the possible immunological or other roles of macrophages in a number of viral infections. In experiments reported here, injection of 30 or 50 mg silica i.p. increased the severity of the oncogenic effects of the murine sarcoma virus (MSV) and Friend virus (FV) in BALB/c mice. Unlike Herpes simplex and Coxsackie B-3 infections, however, passive transfer of adult macrophages to suckling mice did not protect the latter against MSV. In mice injected with silica, histological evidence of the compensatory proliferation of macrophages suggests that precursors of these cells may act as target cells for the virus and that this may override any immunosuppressive response effected by the silica. In addition, there was a considerable enhancing effect on the erythroproliferative response to both MSV and FV by injection of saline 5 h before the virus, and indeed to FV after only a simple abdominal needle puncture. We attributed this to the lymphopenic immunodepressive effects of stress, and our data may explain previously published findings of augmented oncogenic responses in mice after "normal" serum injections. Newborn BALB/c (FV-1b) mice were susceptible to N-tropic FV, but developed resistance by 29 days of age. Antithymocyte serum (ATS) but not silica injections or adult thymectomy ablated this resistance. C57BL (FV-2r) mice were completely resistant to FV; however, those receiving FV and ATS developed late-onset leukaemia histologically characteristic of that produced by the helper component of the FV complex. Images Fig.

Two human lymphoblastoid B-cell lines, WI-L2 and 8866, were infected with the Edmonston strain of measles virus at a multiplicity of infection of 10(-6), and stable persistent infections were established. By immunofluorescence and electron microscopy, the vast majority of cells from both cell lines were expressing viral antigens and releasing virion-like particles. However, very little infectious virus could be detected at 37 degrees C, either by an infectious centers assay or by titration of supernates from persistently infected cultures. When cultures were shifted to 31 degrees C, the cells released a population of virus that was temperature-sensitive. Clonal analysis of supernatant virus at 31 degrees C revealed a highly heterogeneous population of temperature-sensitive mutants, differing in plating efficiency ratios, thermolability, and antigen production at the nonpermissive temperature. Factors such as interferon, defective interfering particles, and extracellular virus do not appear to be important in maintaining the persistent carrier state. These studies have important implications for persistent infections of lymphoid cells in vivo, and the slow neurological diseases associated with measles, subacute sclerosing panencephalitis, and multiple sclerosis.

Summary Peritoneal exudate cells transferred from adult to suckling BALB/c mice before infection of the recipients with Coxsackie B-3 virus protected them against the lethal effects of the virus. Undiluted antibody alone also prevented lethal virus infections in suckling mice but diluted antibody conferred protection only when transferred together with syngeneic adult peritoneal exudate cells. Impairment of macrophage function by intravenous injection of silica increased the susceptibility to virus infection of adult mice. Peritoneal exudate cells inactivated the virus in vitro, and this property may be related to their protective effect in vivo. It is suggested that antibody and host cells collaborate to provide effective resistance against the spread of Coxsackie virus.

Summary Young adult CBA mice developed lethal infections with Coxsackie B-3 virus when immunosuppressed with cyclophosphamide. In the immunosuppressed mice high titres of virus and severe lesions were found in target organs, including the heart and pancreas, as well as persistent viraemia. Immunosuppressed animals showed transient production of IgM but no IgG virus neutralizing antibody; levels of interferon in peripheral blood were higher than in controls. Immunosuppressed mice could be passively protected by administration of serum antibody after infection. The results suggest that circulating antibody plays a critical role in limiting infection of young adult mice by Coxsackie B-3 virus.

The transformation of a potentially neoplastic cell into an autonomous highly malignant and metastatic tumor cell involves a multifactorial cascade of events. This will eventually lead not only to the emergence of a tumor cell with an unlimited potential of replication, but more important will contribute to its ability to ignore and evade homeostatic immune and non-immune regulatory mechanisms. Specifically, those mechanisms which may restrict and direct its growth, dissemination, patterns of differentiation and interaction with the cellular and humoral factors comprising its environment. However, many different factors may contribute to a highly invasive and malignant phenotype. It is obvious that one should expect that a cardinal role should be assigned to alterations in those factors which contribute to the capacity of the malignant cells with its environment at the cell membrane level, which in turn is dependent on the concerted functional expression of specialized membrane associated components (i.e. receptors, cyto adhesion molecules (CAM's), histocompatibility antigens, GAP junction complexes, extracellular matrix components, etc.). In the present studies, we have investigated the contribution of three major factors, which maybe the cause or result of alterations at the level of the cell membrane: MHC encoded antigen expression, susceptibility to the cytolytic activity of NK cells and enhanced expression of the c-K-ras proto-oncogene, as to their development of metastatic capacity of a malignant cell. To address these questions, we used metastatic (IE7) and non-metastatic (IC9) variants of the murine 3-methylcholanthrene induced T-10 fibrosarcoma. Using this system, the following major conceptually important observations were made: A. The restoration by transfection of the expression of membrane associated H-2K encoded glycoproteins abrogates the metastatic capacity of the highly metastatic tumor cell clone, IE7, irrespective of the degree of susceptibility to NK or c-K-ras oncogene expression. This reduction in metastatic capacity is followed by a significant decrease in its tumorigenicity which is concomitant with its ability to induce in vivo potent H-2K restricted CTL's. These results clearly indicate that H-2K region encoded molecules play no apparent role in determining the susceptibility of tumor cells to NK cells, and yet their loss or aberrant expression is a cardinal event in tumor progression towards metastatic capacity, a fact which is supported by similar observations achieved in other murine models (18).(ABSTRACT TRUNCATED AT 400 WORDS)

The transformation of a potentially neoplastic cell into an autonomous highly malignant and metastatic tumor cell involves a multifactorial cascade of events. This will eventually lead not only to the emergence of a tumor cell with an unlimited potential of replication, but more important will contribute to its ability to ignore and evade homeostatic immune and nonimmune regulatory mechanisms. Specifically, those mechanisms which may restrict and direct its growth, dissemination, patterns of differentiation and interaction with the cellular and humoral factors comprising its environment. In the present studies we have investigated the contribution of three major factors which may be the cause or result of alterations at the level of the cell membrane: MHC encoded antigen expression, susceptibility to the cytolytic activity of NK cells and enhanced expression of the c-K-ras proto-oncogene, as to their development of the metastatic capacity of a malignant cell. To address these questions we used metastatic (IE7) and nonmetastatic (IC9) variants of the murine 3-methylcholanthrene-induced T-10 fibrosarcoma. Using this system, the following major conceptually important observations were made: (A) The restoration by transfection of the expression of membrane associated H-2K encoded glycoproteins abrogates the metastatic capacity of the highly metastatic tumor cell clone, IE7, irrespective of the degree of susceptibility to NK or c-K-ras oncogene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute infection of the A-strain mouse neuroblastoma cell line, N 2 A (H-2 a ), with measles virus caused a selective decrease in the expression of H -2 K k determinants, as measured by the quantitative absorption assay. No effect was seen on the H -2 D d determinants. This decrease was paralleled by an increase in expression of measles antigen on the cell surface. Compared to uninfected cells, measles-infected N 2 A cells became less susceptible to lysis by cytotoxic lymphocytes sensitized against H-2 a . The reduced expression of H-2K k was not due to inhibition of total host cell protein synthesis by measles virus, nor was it the result of removal of H-2K k molecules by budding virus. These results are consistent with the concept that an interaction between measles virus and H -2 K k determinants can result in “altered self” antigens, and produce an apparent decrease in the expression of native H -2 K k , but not on H-2D d , antigens suggests that viral antigens may selectively interact with specific histocompatibility antigens but not affect others. The results also demonstrate that altered expression of H -2 determinants is not a sufficient condition for recognition and cytolysis of virus-infected cells by immune T cells.

Susceptibility of adult mice to i.p. infection with HSV-1 was greatly increased by administration of a single dose of cyclophosphamide. Mortality of cyclophosphamide-treated virus-infected mice was associated with increased virus replication and pathologic changes in brain and liver. The development of a fatal infection in immunosuppressed mice could be curtailed after transfer of specifically immune spleen cells. Passively transferred antibody had no such effect. Protective activity of spleen cells was significantly reduced after pretreatment with anti-theta serum. Significant protection was also achieved when normal spleen cells plus immune serum were administered simultaneously. Our results indicate that protection against this virus infection is predominantly T cell dependent, and suggests that antibody-dependent cell-mediated protection may also be operative in vivo.

Measles virus is a ubiquitous human pathogen member of the genus morbillivirus. Acute measles caused by the virus is commonly regarded as a childhood self limited and relatively innocuous disease. However, serious parainfectious complications of measles may occur. In about 1 in 1000 cases, acute encephalomyelitis with perivascular inflammatory and demyelinating damage, may lead to death in 10-20% of patients.1 At least one third of the affected are left with major neurological residua, such as recurrent seizures, mental retardation, hyperkinesis and perceptual disorders.2 In spite of the wide use of live attenuated measles virus vaccine, this exanthem is still a major public health problem.

Natural killer (NK) cells have been implicated in the recognition and killing of a variety of virus infected target cells in vitro, yet their role in vivo remains uncertain. In these experiments, the role of NK cells in the regulation of resistance to herpes simplex virus-1 (HSV-1) was studied. Adult C57BL/6 mice are resistant to HSV-1 (HFEM strain), but are rendered highly susceptible by treatment with cyclophosphamide 24 hr prior to infection. In this model, passive transfer of 10(8) normal spleen cells or 10(7) poly I:C-treated spleen cells provided protection for 72% of the recipients. Spleen cells from NK cell-deficient beige mice similarly treated failed to engender passive protection. The phenotype of the cells responsible for transferring protection was NK1.1+, and asialo GM1+. Transfer of NK cells resulted in marked reduction of HSV titers in the livers and brains of recipients. These experiments provide direct evidence for a role for NK cells in protection against development of fatal HSV infection in mice.

Infants suffering from bronchopulmonary dysplasia (BPD) are known to have low levels of vitamin A, a factor which may be implicated in the pathogenesis of the condition. The ability of retinoic acid (RA) (one of the active forms of vitamin A) to influence the production of superoxide anion (02-) and hydrogen peroxide (H2O2) by stimulated human adult or cord blood neutrophils and macrophages has been studied. RA was found to inhibit the O2- and H2O2 production in a dose-dependent manner. The time required for maximal inhibition was 30 min for neutrophils and 24 h for macrophages. Although cord blood neutrophils produced larger quantities of O2- and H2O2 both with and without RA, the degree of inhibition was similar in both adult and neonatal cells (40-60%). The results suggest that retinoic acid may prevent neutrophil and macrophage mediated lung damage by inhibiting the production of toxic oxygen compounds, especially in BPD conditions.

From the studies summarized here a complex picture of the role played by MHC products in determining tumorigenicity and metastasis is emerging. In order to be able to understand this relationship better, it is necessary to consider several factors. 1. Each tumor system or neoplastic tissue is unique, and its behavior reflects the influence of cell-specific characteristics, as well as its ability to modulate other cells and tissues--including cells belonging to the immune system--and also to be modulated by other cells and soluble factors. 2. Since metastasis formation is a multistep process in which only small subpopulations of tumor cells with complex and defined phenotypes are able to colonize secondary tissues, elimination of even one single phenotypic component of this structured process can easily reverse the metastatic capacity of the cells. Acquisition of metastatic ability, on the other hand, would be a more difficult task, since any new characteristic expressed by the cells or induced experimentally, such as gene transfection or results of IFN treatment, must be expressed in a temporal manner and in concert with other cellular characteristics. Therefore, an experimental protocol measuring a specific element in determining metastasis can easily produce conflicting results, depending on the type of cells and genetic background of the host studied. 3. The level of specific MHC products on tumor cells is one among many other cell characteristics that may determine the metastatic potential of cells. Moreover, each of the class 1 MHC products, and the relationship among them, including other than the classical K, L, or D products (Brickell et al., 1983), should be regarded as independent entities, with possible different regulatory roles in cell-cell recognition, in a general sense, which may be involved in determining invasiveness and homing as well as recognition by the immune system. 4. Both specific T-cell and nonspecific natural mediated immunity (which is much less understood) are involved in the selection of the metastatic cell population. 5. Immunogenicity of tumors is not necessarily determined by high levels of MHC antigen expression; it is also dependent on the level of TSA. Thus, immunoselection mediated by T lymphocytes during metastasis formation could be directed against both MHC and TSA antigens. Therefore, low expression of MHC antigens by metastatic cells as a result of immunoselection is not always observed.(ABSTRACT TRUNCATED AT 400 WORDS)

The role of natural killer (NK) cells in retrovirus-induced leukemogenesis was studied. These cells which do not require prior sensitization are considered as a part of the body's defense system against tumor development and spread. Neonate BALB/c mice infected with Moloney murine leukemia virus (MoLV) develop leukemia within 3-6 months. The MoLV-infected mice showed a progressive loss of endogenous and augmented NK activity, correlated with the development of the leukemic state. Mixing of spleen cells from tumor-bearing mice with NK-augmented splenocytes resulted in suppression of NK activity. In addition, mixing of T cell lines isolated from MoLV-induced tumors with augmented splenocytes also resulted in the down-regulation of NK cell activity. The present study demonstrates that tumor cells from leukemic organs and leukemic T cell lines can actively suppress NK cell function. It is postulated that after MoLV infection the progression of virus-transformed T cells to a fully developed tumor depends on the ability of these cells to down-regulate NK cell activity and thus escape immune surveillance.

Cultures of phytohemagglutinin-stimulated, unstimulated, and frozen and thawed mononuclear (as controls) cells from normal human donors were infected with different viruses. A variable pattern of virus infectivity was noted, and influenza and Coxsackie virus were rapidly inactivated even by stimulated lymphocytes. Direct inactivation by lymphocytes may be one form of host defense in infection by some viruses.

Clone NS20Y of the mouse neuroblastoma C1300 was infected with wild-type Edmonston measles virus, and, after a transition to a carrier culture, became persistently infected. Persistently infected clones were derived and characterized morphologically by the appearance of multinucleate giant cells and nucleocapsid matrices in cytoplasm and nucleus, but very few budding virus particles. Antimeasles antibodies markedly suppressed the expression of viral antigens and giant cells, and the effect was totally reversible. When the cells were cultured at 33 degrees C, the number of giant cells began to diminish and ultimately disappeared; in contrast, when cultured at 39 degrees C, the cultures invariably lysed. Yields at 33 degrees C were ca. 2 logs lower than those at 39 degrees C. Cells cultured at 33 degrees C produced relatively high levels of interferon, whereas those at 39 degrees C produced little or no interferon. When the persistently infected cultures were exposed to anti-interferon alpha/beta serum at a nonpermissive temperature, there was a marked increase in multinucleate cells, suggesting that maintenance of the persistence state and its regulation by temperature may be related to the production of interferon. Viral isolates from cells cultured at 39 degrees C were obtained, and 90% of viral clones were found to be cold sensitive. Complementation studies with different viral clones indicated that the cold-sensitive defect was probably associated with the same genetic function. Western blot analysis of the persistently infected cells indicated a significant diminution and expression of all measles-specific proteins at a nonpermissive temperature. Infection of NS20Y neuroblastoma cells with the cold-sensitive virus isolates resulted in the development of an immediate persistent infection, whereas infection of Vero or HeLa cells resulted in a characteristic lytic infection, suggesting that the cold-sensitive mutants may be selected or adapted for persistent infection in cells of neural origin.