Chronic ethanol consumption disturbs G-protein expression and inhibits cyclic AMP-dependent signaling in regenerating rat liver. Evidence suggests that ethanol desensitizes hepatocytes to the trophic effects of hormones. Cyclic AMP-dependent signals are important regulators of intermediary metabolism, cellular proliferation and differentiation, and modulate liver growth during hepatic regeneration. The events leading to cyclic AMP accumulation after partial hepatectomy were characterized in rats consistently fed ethanol-containing diets and compared with results in rats fed isocaloric amounts of nonethanol diet to determine whether altered cyclic AMP-dependent signal transduction contributes to ethanol-associated aberrations in hepatic growth regulation. Ethanol treatment significantly inhibited hepatic accumulation of cyclic AMP after partial hepatectomy. This was most likely the result of decreased synthesis of cyclic AMP because activation of adenylyl cyclase by agents acting through receptors (e.g., glucagon or isoproterenol), GTP-binding proteins (GTP-gamma-S) and directly on adenylyl cyclase (e.g., forskolin) was significantly inhibited in ethanol-fed rats. Both homologous and heterologous desensitization contributed to this effect. beta 1-Adrenergic receptors were relatively down-regulated 6 hr after partial hepatectomy in ethanol-fed rats, whereas glucagon receptor kinetics were similar in the two groups. Liver membrane expression of GTP-binding proteins differed markedly after partial hepatectomy in ethanol-fed and pair-fed rats. Ethanol significantly inhibited post-partial hepatectomy induction of the stimulatory G protein, Gs alpha but led to overexpression of the inhibitory, G(i)2 alpha, subunit. Steady-state messenger RNA levels of these G proteins were similar in ethanol-fed and pair-fed rats, suggesting that ethanol inhibits G protein expression posttranscriptionally.(ABSTRACT TRUNCATED AT 250 WORDS)


Interaction of bile salts with calcium hydroxyapatite: inhibitors of apatite formation exhibit high-affinity premicellar binding. Of the major human bile salts, only the glycine-conjugated dihydroxy species prevent the transformation of amorphous calcium phosphate to calcium hydroxyapatite, a component of gallstones; we have proposed that this inhibition occurs by competition between the bile salt and HPO4(2-) anions for binding site on the apatite crystal embryo. Now we show that the binding affinity of bile salts to fully mature hydroxyapatite has the following order: glycine-conjugated dihydroxy salts > taurine-conjugated dihydroxy salts > glycocholate approximately taurocholate. Glycine-conjugated dihydroxy bile salts bound with high affinity as "premicellar" aggregates, but the remaining species appeared to bind as a wider range of aggregate sizes. Glycochenodeoxycholate binding was decreased as the pH increased from 6.6 to 9.8 and the apatite surface charge reversed from net positive to net negative. Binding was competitively inhibited by HPO4(2-), but not by H2PO4-. Ca2+ promoted the binding of glycochenodeoxycholate, taurochenodeoxycholate and glycocholate, and for the latter two bile salts the increase was associated with enhanced "premicellar" binding. The binding of taurocholate was not influenced by Ca2+. When either glycocholate or taurocholate was mixed with glycochenodeoxycholate, mixed aggregates were formed that had a lower affinity for apatite than had pure glycochenodeoxycholate aggregates. Because only glycine-conjugated dihydroxy bile salts inhibit apatite formation, these results suggest that inhibition depends on high-affinity "premicellar" bile salt-apatite binding.


Effect of case management on rehospitalization and utilization of ambulatory care services. To explore whether intensive case management would reduce discharged psychiatric inpatients' rates of rehospitalization and increase their use of outpatient ambulatory care services, the authors assigned 435 patients discharged from a public general hospital in New York City to three types of postdischarge services: multidisciplinary intensive outreach case management, less intensive Community Support System (CSS) case management services, or routine aftercare but no case management. Patients eligible for CSS were generally more impaired and had more extensive histories of psychiatric hospitalization. At follow-up 18 to 52 months after entering the study, patients who received intensive outreach case management had twice as many psychiatric hospitalizations as the routine aftercare group and had a pattern of utilization of hospital-based ambulatory care services similar to the more impaired CSS group. Intensive outreach case management did not appear to stimulate sufficient use of ambulatory care services to reduce patients' frequency of psychiatric hospitalization.


Cytomegalovirus infection of the human placenta: an immunocytochemical study. In congenital cytomegalovirus (CMV) infection histologic evaluation of the placenta is often unrevealing. In the present study immunocytochemistry to CMV immediate early and early nuclear antigens was used to characterize placental involvement in six cases of symptomatic intrauterine CMV infection. Histologic examination had demonstrated diagnostic viral inclusions in one placenta and non-specific villitis in another. However, immunocytochemistry revealed CMV infection in five of the six placentas, including three with no pathologic changes on routine histologic evaluation. Infected cells were located primarily in the villous stroma. In one case immunoperoxidase staining showed infection in the syncytiotrophoblast. Infected endothelial cells were demonstrated by double staining for CMV and factor VIII antigen. No double-stained cells were seen in tissue sections stained for CMV immediate early nuclear antigen or the human macrophage-associated CD68 antigen, which is expressed in Hofbauer cells. In conclusion, specific immunoperoxidase staining was more sensitive for demonstrating placental CMV infection than was histologic examination and it aided in the characterization of infected cells.


Immunohistochemical analysis of small cell tumors of the thyroid gland: an Eastern Cooperative Oncology Group study. The majority of small cell anaplastic tumors of the thyroid gland are generally believed to be non-Hodgkin's lymphomas, including most of those formerly classified as small cell carcinomas. Using a panel of antibodies capable of detecting epithelial, neuroendocrine, and B and T cells in paraffin-embedded tissue sections, we studied 68 thyroid neoplasms in which the original diagnosis was small cell carcinoma or lymphoma. Sixty-three of the tumors were identified as lymphomas of B-cell origin on the basis of L26 reactivity used in conjunction with light chain restriction and MB2 immunostaining. Two additional tumors were classified as lymphomas of indeterminate phenotype. Immunophenotyping indicated an epithelial origin in the remaining three tumors. No cases of medullary carcinoma were detected by immunostaining. Histologic review revealed a predominance of large cell and immunoblastic lymphomas, with low-grade lymphomas of mucosa-associated lymphoid tissue histology accounting for only five cases. Our findings indicate that the majority of small cell anaplastic tumors of the thyroid are B-cell lymphomas. Although primary small cell carcinoma of the thyroid may rarely occur, this diagnosis should not be made without immunohistologic confirmation.


Biologic characteristics of specific human papillomavirus types predicted from morphology of cervical lesions. Human papillomavirus (HPV) DNA was detected by Southern blot hybridization in cervicovaginal lavage samples from 199 of 329 (60.5%) women attending a municipal hospital colposcopy clinic. Human papillomavirus was identified in 195 of 264 (73.9%) patients with a squamous intraepithelial lesion or cancer on biopsy or Papanicolaou smear (Bethesda system) compared with 11 of 65 (16.9%) without squamous intraepithelial lesion (P < .0001). The most common HPV type identified was HPV 16 (20.6% of positive samples), and 36.7% of isolates contained uncharacterized HPVs. Of women with cervical intraepithelial neoplasia (CIN) grade III or cancer, 23.4% were infected with HPV 16 compared with less than 4% with any other single HPV type. Based on biopsy diagnosis in patients infected with specific HPV types, HPVs 6 and 11 had low oncogenic potential; HPVs 18, 31, 35, and 45 had intermediate oncogenic potential; and HPVs 16 and 33 had high oncogenic potential. Hyperchromatic, unusually enlarged nuclei ("meganuclei"), and/or abnormal mitoses were found significantly more often in lesions infected with HPVs 16, 33, and 35 than in those infected with HPVs 6, 11, 18, 31, and 45, even in low-grade lesions, and may represent a histologic marker for HPVs with significant oncogenic potential. Human papillomavirus capsid protein was detected significantly less often by immunocytochemical staining in CIN I and CIN II lesions infected with HPVs 16 and 33 (8.3%) than in those infected with HPVs 6, 11, 18, and 31 (60%; P = .007), suggesting early abnormalities in cellular differentiation in lesions infected with highly oncogenic HPVs.


Cytogenetic and pathologic aspects of Ewing's sarcoma and neuroectodermal tumors. Diagnostic classification of poorly differentiated, round cell, primitive neuroectodermal neoplasms, including Ewing's sarcoma, peripheral neuroepithelioma, Askin's tumor, and esthesioneuroblastoma, is challenging to the surgical pathologist using conventional histopathologic approaches because of very similar and overlapping morphologic and cytologic features. Furthermore, distinguishing these neoplasms from neuroblastoma, embryonal rhabdomyosarcoma, small cell osteogenic sarcoma, and non-Hodgkin's lymphoma can be difficult. This paper describes and reviews the cytogenetic and molecular genetic changes in these tumors and demonstrates how the ability to detect these changes has enabled a greater understanding of the histogenesis, classification, diagnosis, and prognosis of these neoplasms.


A double blind study of single dose azithromycin and doxycycline in the treatment of chlamydial urethritis in males. To compare the efficacy and safety of azithromycin and doxycycline in the treatment of males with uncomplicated urethritis caused by chlamydia trachomatis. A multicentre, double-blind, randomised treatment study. 130 male outpatients with clinical signs and symptoms of urethritis. STD clinics at four Norwegian University Hospitals. Patients were randomly allocated to 1000 mg azithromycin as single dose or doxycycline 100 mg twice daily for 7 days. Clinical, bacteriological and safety assessments were made at entry and after 1 and 2 weeks. Safety data were also repeated after 4 weeks. Demographic data were similar in both groups. At the week 1 assessment bacteriological eradication was achieved in 44 of 44 evaluable azithromycintreated patients and in 42 of 42 in the doxycycline group. At the week 2 assessment the corresponding figures were 35 of 35 and 34 of 34 respectively. Azithromycin 1000 mg single dose was as effective as doxycycline 100 mg twice daily for 7 days in male patients with chlamydial urethritis.


Fluorescence in situ hybridization mapping of human chromosome 19: mapping and verification of cosmid contigs formed by random restriction enzyme fingerprinting. Automated restriction enzyme fingerprinting of 7900 cosmids from chromosome 19 and calculation of the likelihood of their overlap based on shared fragments have resulted in the assembly of 743 sets of overlapping cosmids (contigs). We have mapped 22% of the formed contigs (n = 165) and all of the contigs with minimal tiling paths exceeding 6 members (n = 50) to chromosomal bands by fluorescence in situ hybridization using DNA from at least one member cosmid. The estimated average size of the formed contigs is 60-70 kb. Thus, members of a correctly formed contig are expected to lie close to each other in metaphase and interphase chromatin. Therefore, we tested the contig assembly process by comparing the band assignment of two or more members selected from each of 97 contigs. Forty-two of these contigs were further characterized for valid assembly by determining the proximity of members in interphase chromatin. Using these tests, we surveyed a total of 431 joins counted along the minimal tiling path (280 in interphase as well as metaphase) and found 6 erroneous joins, one in each of 6 contigs (6% of tested).


Genomic organization of the human folate receptor genes on chromosome 11q13. There has been interest in the high affinity folate receptor (FOLR) recently because of its high expression in the majority of ovarian tumors. The FOLR genes are part of a family that includes an adult gene, a fetal gene, and one or more pseudogenes, which have been localized to chromosome 11. As a step toward understanding why the adult FOLR gene product is expressed on tumors, we have determined the organization of all the human FOLR-related genes. YAC clones were isolated using the adult FOLR probe. The organization of the locus was determined by PFGE of YAC DNA and by YAC fragmentation. Four FOLR-related genes were found within 140 kb. The adult and fetal genes are not more than 23 kb apart, with the 3' end of the adult gene facing the 5' of the fetal gene. A physical map of over 900 kb of the surrounding region was also constructed. The chromosomal assignment of the FOLR locus was refined to 11q13.3-q13.5 telomeric of the FGF3 locus using fluorescence in situ hybridization.


Exclusion of linkage between hypokalemic periodic paralysis (HOKPP) and three candidate loci. Hypokalemic periodic paralysis (HOKPP) is an autosomal dominant neuromuscular disorder characterized by flaccid paralysis accompanied by lowered serum potassium levels. We have tested polymorphic markers linked to the adult skeletal muscle sodium channel (SCN4A) locus at 17q23-q25, the T-cell receptor beta (TCRB) locus at 7q35, and the H-Ras cellular proton-cogene locus (HRAS) at 11p15.5 for linkage with the affected phenotype in a single multigenerational pedigree. No evidence for genetic linkage to HOKPP was found at any of the candidate loci.


Frequent polymorphism in the 13th exon of the adenomatous polyposis coli gene. We have studied the DNA from 170 individuals affected with familial adenomatous polyposis and from 20 uneffected individuals. Denaturing gradient gel electrophoresis of polymerase chain reaction amplified DNA from the adenomatous polyposis coli (APC) gene demonstrated three major patterns consistent with the existence of two different sequences in exon 13 of the gene in the human population. Direct sequencing of the amplified product from DNAs producing these three patterns confirmed the presence of a polymorphism in the coding region of the APC gene.


The QM gene is X-linked and therefore not involved in suppression of tumorigenesis in Wilms' tumor. Inactivation of one or more tumor-suppressor genes on the short arm of chromosome 11 is thought to play a role in the etiology of Wilms' tumor. A candidate gene, QM, was recently isolated by subtractive hybridization between a tumorigenic cell line (deleted for part of 11p) and a non-tumorigenic cell line (the tumorigenic cell line carrying an extra t(X;11)copy). We show here with an exon-specific polymerase chain reaction that the genomic homolog of the QM cDNA is located in the G6PD-color vision genes region in Xq28. No homologous sequences could be detected on 11p. Our experiments indicate that the QM gene is not involved in the suppression of Wilms' tumor.


The human ubiquitin/52-residue ribosomal protein fusion gene subfamily (UbA52) is composed primarily of processed pseudogenes. Two members of the human ubiquitin gene subfamily encoding ubiquitin fused to a 52-residue ribosomal protein (UbA52) were cloned, sequenced, and analyzed. Both were found to be processed (reverse-transcribed) pseudogenes, containing several nucleotide substitutions and deletions that severely disrupt their potential open-reading frames. Genomic hybridization analysis using probes derived from a functional UbA52 gene (UBA52) suggests that the UbA52 subfamily consists of one intron-containing, transcriptionally active gene and approximately eight processed pseudogenes, a feature characteristic of mammalian ribosomal protein gene families.


A human dimorphism resulting from loss of an Alu. The molecular phylogeny of Alu and other repeated sequences in the human genome provides clues to events during primate evolution. A subclass of human Alu's has been previously identified as dimorphic insertions within members of the medium reiteration frequency (mer) class of repeats, reflecting the complicated sequence of insertion and radiation events leading to the current human genome structure. One dimorphic Alu is located within a previously unidentified mer family member, in the first intron of the human T4 (CD4) gene. The insertion (Alu+ allele) has a frequency of approximately 70% in Europeans and Africans and is homozygous in 20 Asian samples. Polymerase chain reaction amplification, direct DNA sequencing, and Southern analysis using oligonucleotide probes revealed that the Alu- allele was derived from the Alu+ allele by loss of part of the inserted sequence. Comparison with a tightly linked marker within the human genome and studies of baboon DNA samples revealed that the original insertion was a relatively early event in primate evolution, but that the Alu sequence loss leading to the dimorphism has occurred much more recently. Loss of Alu insertions therefore represents one mechanism for the generation of human Alu dimorphisms.


DNA rearrangements in the alpha 5(IV) collagen gene (COL4A5) of individuals with Alport syndrome: further refinement using pulsed-field gel electrophoresis. Alport syndrome (AS), an X-linked kidney disorder, has been shown to be caused by mutations in the gene for the alpha 5-chain of type IV collagen (COL4A5), which maps to Xq22. On the basis of the results of conventional Southern blot analysis of AS patient DNAs, we employed pulsed-field gel electrophoresis to characterize further three gene rearrangements at the 3'-end of alpha 5(IV). We were able to construct long-range restriction maps for all three of these patients and deduce the extent and nature of each rearrangement. One of these mutations is a 450-kb simple deletion that includes 12 kb of the alpha 5(IV) gene. A second mutation has been shown to be a direct duplication of 35 kb of alpha 5(IV) genomic DNA, and a third mutation involves a complex insertion/deletion event resulting in an overall loss of 25 kb.


Syntenic assignments of visual transduction genes in cattle. To establish syntenic relationships of phototransduction genes, we have mapped the genes encoding the alpha-, beta-, and gamma-subunits of rod cGMP phosphodiesterase (PDE) (PDEA, PDEB, PDEG), the alpha'-subunit of cone PDE (PDEA2), and the rod cGMP-gated channel (CNCG) to bovine syntenic groups. The rod cGMP PDE alpha-, beta-, and gamma-subunit genes map to bovine syntenic groups U22, U15 (chromosome 6), and U21 (chromosome 19), respectively. The rod cGMP-gated channel gene also maps to syntenic group U15, and the bovine cone alpha'-subunit gene maps to U26 (chromosome 26). With the exception of the cone PDE alpha'-subunit gene, which has not been mapped in other mammals, all of these genes have been assigned to conserved chromosomal regions shared among bovine, human, and mouse. A compilation of currently known syntenic assignments and predictions regarding future assignments of phototransduction genes in human, mouse, and cattle is presented.


Confirmation of the localization of the human GABAA receptor alpha 1-subunit gene (GABRA1) to distal 5q by linkage analysis. The GABAA receptor is the major inhibitory neurotransmitter receptor in the mammalian brain. To date, 14 genes that encode subunits of this receptor have been identified; these appear to be scattered throughout the human genome and are under investigation as candidate loci for a number of neurological and psychiatric disorders. We report here a highly polymorphic (dC-dA)n repeat within the human alpha 1-subunit gene (GABRA1). Typing of this marker in the Centre d'Etude du Polymorphisme Humain (CEPH) panel of families confirms the previous assignment of the GABRA1 locus to the distal portion of chromosome 5q by demonstrating linkage to the markers CRI-L45 (D5S61) (Zmax = 11.00, theta max = 0.15), CRI-V1022 (D5S54) (Zmax = 7.25, theta max = 0.20), and CRI-P148 (D5S72) (Zmax = 5.71, theta max = 0.24).


Molecular approaches to the study of human B-cell and (auto)antibody repertoire generation and selection. We have shown that the restricted repertoire of VH genes expressed in second trimester human fetal liver is not solely determined by JH proximity. Furthermore, by following the fate of two VH gene segments in different B-cell repertoires, we have provided evidence that multiple factors contribute to the frequency with which individual VH genes are utilized. We found that the repertoire of adult blood IgM-bearing B cells contains a high proportion of B lymphocytes that express extensively mutated VH genes. Finally, we show that somatically-mutated variants of particular VH and VL genes that, in germline configuration, are frequently found in the early B-cell repertoire and in natural autoantibodies, encode pathogenic IgG autoantibodies characteristic of human SLE. These VH and VL genes harbor all the characteristics of an antigen-driven B-cell activation and selection process.


Spectroscopic probe of distribution of the non-intercalating drug netropsin between DNA and heparin. Circular dichroism has been used as a monitoring tool to probe the distribution of the non-intercalating drug netropsin (NTPS) between the two biomolecules DNA and heparin. The stoichiometry of the interaction of the individual biomolecules and the drug is determined from conductometric titrations; the titration in each case shows two breaks corresponding to two stoichiometries of interaction. Though netropsin is non-intercalating, DNA wins over heparin in binding the drug due to strong hydrogen bonding capability of NTPS in the minor grooves of DNA through its greater than NH donor groups. Potential hydrogen bond breakers like KF and urea reduce the induced dichroism of NTPS-DNA system, probably dislodging some drug from DNA through hydrogen bond breaking.


Silanized silica bound trypsin as analytical probe. Trypsin immobilized by covalent coupling to silanized silica shows significant activity (30-38%) and greater thermostability as compared to soluble trypsin. Proteolytic processing of albumin at varying periods suggest that the enzyme matrix can be used efficiently for limited proteolysis. Repeated use of the immobilized enzyme in protein digestion produces similar products as seen by electrophoretic analysis. Also, digestion of albumin by the immobilized enzyme follows similar pattern as that by soluble enzyme. The enzyme matrix can be easily removed from the incubation mixture. The results indicate the possibility of the immobilized enzyme for its effective application as analytical tool in peptide mapping and limited proteolytic processing.


Interleukin-2 may enhance or inhibit antibody production by B cells depending on intracellular cAMP concentrations. The primary IgM response of murine B lymphocytes against red blood cell-bound antigens can be induced by incubating antigen-reactive B cells either with the lymphokines interleukin-1 (IL-1) and IL-2 together with the nucleoside cAMP, or by the addition of antigen-specific helper T cells. The reactivity of B cells is strongly influenced by the T-cell lymphokine IL-2. IL-2 inhibits the cyclic adenosine 3',5'-phosphate (cAMP)-dependent B-cell response when it is allowed to act on the cells prior to cAMP. On the other hand, if IL-2 acts on B cells together with or after cAMP, it synergizes with the nucleoside and enhances the immune response. A similar effect of IL-2 is observed in the T-cell-mediated activation of B cells. If IL-2 is present before helper T cells interacted with B cells, it inhibits antibody production. The inhibitory IL-2 effect is reversed by the simultaneous addition of exogenous cAMP. The finding supports the hypothesis that Ia ligation by T cells results in B cells in the elevation of cAMP which acts as an important second messenger in B cells. The antagonism between cAMP and IL-2 was also examined in the pre-B-cell line 70Z/3. The nucleoside is highly toxic to 70Z/3 pre-B cells and a majority disintegrates within hours of exposure to the nucleoside. The surviving cells undergo phenotypic differentiation expressing surface Ig kappa chains and major histocompatibility complex (MHC) class II molecules, and increase the expression of IL-2 receptor (R). The phenotypic differentiation requires the presence of IL-1. IL-2 inhibits both of these B-cell responses to cAMP, the IL-1-independent cell death, and the IL-1-dependent phenotypic differentiation.


The comparative role of 1,25-dihydroxycholecalciferol and phorbol esters in the differentiation of the U937 cell line. The active metabolite of cholecalciferol, 1,25-dihydroxycholecalciferol (1,25-DHCC), is a mononuclear phagocyte product with immunoregulatory properties which can influence not only surrounding T cells but also other mononuclear phagocytes; and which acts in an autocrine fashion. In this study we have used the U937 cell line as a starting point model to investigate further the comparative role of 1,25-DHCC and phorbol myristate acetate (PMA) upon growth, differentiation and phenotype in the mononuclear phagocyte system, and have correlated our findings with changes in 1,25-DHCC metabolism and receptor expression. Both 1,25-DHCC and PMA inhibit growth and differentiate U937 cells in a dose-dependent fashion. When used together, however, at low doses of PMA, 1,25-DHCC protects against the PMA-induced growth inhibition. At high concentrations of both compounds there is a decrease (1,25-DHCC) or an increase (PMA) in 1,25-DHCC receptor expression, with either 24-OHase (1,25-DHCC) or 1-OHase (PMA) synthesis. If the compounds are used in combination the receptor levels are equivalent to controls, and both enzymes are produced. The phenotype of the 1,25-DHCC-induced cells shows light adherence, class I+, increase in CD4 and CD14 and decrease in CD71. The PMA-induced cell is tightly adherent, class I+, and strongly positive for CD13 with a concomitant decrease in both CD4 and CD71. These findings suggest another role for 1,25-DHCC in the mononuclear phagocyte system, as a potential mitogenic agent. They also suggest that 1,25-DHCC may act at both membrane and nuclear levels within this model of the mononuclear phagocyte pathway and demonstrate one possible way in which physiological peripheral macrophage heterogeneity might be induced, i.e. due to the nature of the signals which are implicated during differentiation. The presence of increased CD4 and decreased CD13 on the surface of 1,25-DHCC-differentiated cells, and vice versa on PMA-differentiated cells, illustrates how this may then be reflected in functional mononuclear phagocyte heterogeneity, which may in turn be reflected in differential peripheral function.


Restimulated memory Tc cells have a higher apparent avidity of interaction with targets than primary virus-immune Tc cells as indicated by anti-CD8 blocking. Previous experiments have shown that whereas a secondary in vitro Kunjin-immune cytotoxic T (Tc) cell population lysed equally well targets infected with either native flavivirus or a recombinant vaccinia virus expressing the immunodominant determinant, primary in vivo Kunjin-immune Tc cells were able to lyse only the recombinant vaccinia virus-infected targets. Using CD8 blockade to assess the avidity of T cell-target interaction, recombinant-infected targets express antigen more efficiently than native flavivirus infected targets and secondary in vitro Kunjin-immune Tc cells have a higher avidity for targets than do primary in vivo Kunjin-immune Tc cells. Secondary in vivo influenza-immune Tc cells are also of higher avidity than primary in vivo influenza-immune Tc cells. Thus, a restimulated memory Tc cell population interacts with targets with greater avidity than does a recently activated naive population.


Activation of human macrophages for the killing of intracellular Trypanosoma cruzi by TNF-alpha and IFN-gamma through a nitric oxide-dependent mechanism. The protozoan parasite Trypanosoma cruzi is able to replicate in the cytoplasm of primary resident macrophages, but is killed by activated macrophages. Pretreatment of human macrophages with recombinant IFN-gamma and to a lesser extent with TNF-alpha, induced a significant trypanocidal activity. Furthermore, TNF-alpha had a synergistic effect with IFN-gamma on macrophage activation in T. cruzi killing. Similarly, IFN-gamma triggered the production of nitric oxide (NO) by macrophages, whereas TNF-alpha was less effective, although it was also synergistic with IFN-gamma. Both NO production and trypanocidal activity, but not superoxide (O2-) generation, induced in macrophages by TNF-alpha or IFN-gamma alone or in combination, were inhibited by N-monomethyl-L-arginine (N-MMLA), a competitive inhibitor of NO synthase activity. Furthermore, a strong correlation was found between the levels of NO production and trypanocidal activity induced by different lymphokine preparations. These results suggest that IFN-gamma and TNF-alpha are involved in the activation of the trypanocidal activity of human macrophages through a NO-dependent mechanism.


Selective regulation of antigen-specific IgE response by cyclic AMP level in murine lymphocytes. We have reported that prostaglandin E2 (PGE2) is a selective stimulator of the antigen-specific IgE response [6]. Because PGE2 is known to elevate intracellular cAMP, we investigated the regulatory role of cAMP in the production of antigen-specific IgE. Anti-TNP IgE response was induced by stimulating TNP-KLH-primed BALB/c spleen cells with the same antigen in vitro. Addition of 10-100 microM dibutyryl cAMP (DBcAMP) to the lymphocyte culture resulted in a 2-3-fold increase in anti-TNP IgE response without affecting the production of anti-TNP IgG1 or IgM. Forskolin, a stimulator of adenylate cyclase, also specifically augmented the IgE response. In contrast, 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, suppressed IgE production in an isotype-specific manner. These results suggest that IgE synthesis can be selectively modulated by intracellular cAMP level. Enhancement of IgE production by DBcAMP was observed, particularly in highly primed spleen cells, suggesting that IgE-committed B cells are subjected to regulation by cAMP.


Antibodies against free radical modified native DNA recognize B-conformation. Hydroxyl radical, a prominent entity of reactive oxygen species, is known to modify cellular DNA and has been implicated in several human diseases. In the present studies, the radical was generated by exposure of hydrogen peroxide to 254 nm light in the presence of native calf thymus DNA. Single strand breaks, decrease in Tm and modification of adenine and thymine were some of the modifications observed in nDNA. Antibodies induced in experimental animals against the modified DNA were immunogen specific. These antibodies also recognize native B-conformation. It was observed that naturally occurring anti-native DNA autoantibodies from SLE sera recognize modified DNA in direct binding and competition ELISA. Gel retardation assay reiterated the formation of immune complexes between induced antibodies and DNA fragments of around 300 bp (B-conformation). The possible significance of these findings in the etiology of SLE has been discussed.


Intrahepatic cholangiocarcinoma: report of a surgically resected case. We report a middle aged female with intrahepatic cholangiocarcinoma which was resected successfully. Because of an unusual histology, a microscopic diagnosis could not be established pre-operatively. The tumor recurred locally 21 months after surgery. The case is reported mainly for the diagnostic problems it created.


Hybridization methods other than PCR: an update. To detect the presence of HPV DNA in samples of differing origin, a variety of hybridization methods are available. The advantages of each of these methods are discussed. Recent improvements to certain methods, that increase the value of their practical application, are highlighted.


Polymerase chain reaction-based methods for the detection of human papillomavirus DNA. The unprecedented sensitivity of the polymerase chain reaction (PCR) DNA amplification technology provides numerous advantages for the detection of human papillomavirus (HPV) DNA. In this chapter, the principles of the method and its advantages are discussed; disadvantages of the technology and proposed solutions are also presented. To accommodate the complexity of the many distinct HPV types, both type-specific and consensus PCR-based detection methods have evolved. Consensus primer systems, which afford the amplification of a broad spectrum of HPV types, have proven particularly useful in epidemiological studies and in novel virus identification. Since the introduction of PCR into the HPV field, there have been rapid advances and improvements PCR technology. However, standardization of PCR methods for HPV detection is essential for research to fully benefit from the technology. To that end, specific guidelines regarding experimental controls, assessment of analytic sensitivity and data interpretation are outlined here.


Detection of human papillomavirus DNA without amplification: prospects for clinical utility. Certain HPV types are major risk factors for the development of cervical and other human cancers. This chapter describes some of the problems which have precluded accurate diagnosis of HPV-related conditions in many studies. Despite the confusion, a multitude of reports demonstrate that HPV DNA testing has clinical utility, and future investigations should be directed at more accurately delineating its role in human health care.


Serological assays for the detection of HPV antibodies. A variety of serological assays to detect antibodies to genital-type HPVs have been developed. Bacterially expressed fusion proteins, synthetic peptides and HPV 11 virus propagated in a xenograft system have been the most commonly used antigen targets in either Western blot assays or ELISAs. HPV antibodies have been readily detected and most studies suggest that they are type-specific. Primarily, antibodies appear to be directed against the capsid antigens. The presence or titre of antibodies to the HPV 16 E7 protein is strongly associated with cervical cancer in approximately 25% of cases. The significance of antibodies to other HPV antigens, or of antibodies which recognize conformational epitopes is less clear. Attempts to validate the sensitivity and specificity of serological assays are extremely preliminary, and are complicated by a lack of understanding of the natural history of papillomavirus infections.


Validation of hybridization assays: correlation of filter in situ, dot blot and PCR with Southern blot. A number of validation experiments have compared the most commonly used HPV hybridization methods with the accepted gold standard, Southern blot hybridization. The methods discussed are filter in situ hybridization (FISH), dot blot hybridization (ViraPap/ViraType), and polymerase chain reaction (PCR). FISH now appears to be too inaccurate to be recommended for future epidemiological studies. ViraPap/ViraType compares well to Southern blot, but is limited to the detection of seven genital HPV types. PCR-based methods may be more sensitive than Southern blot and are likewise capable of detecting most known genital HPV types. Direct comparisons of the several available PCR methods should be performed. Currently, there is no perfect method for HPV testing, because Southern blot itself is prone to some errors in performance and interpretation. Given that the scientific and clinical usefulness of HPV tests depends on the repeatability and accuracy of the assays, more intra- and inter-assay comparisons should be done to establish reference standards applicable to this area of molecular diagnostics.


Measurement errors in epidemiological studies of human papillomavirus and cervical cancer. Measurement errors have been an important concern in studies of human papillomavirus (HPV) and anogenital cancers. Misclassification of HPV infection status is a possible explanation for incoherent findings in previous epidemiological studies purporting to show an etiological role for HPV in cervical cancer. Even low levels of misclassification of HPV infection can cause severe underestimation of HPV prevalence in field surveys, bias the association between HPV and sexual activity, and impair the ability to control statistically the relation between sexual activity and neoplasia by viral status. The present report focuses on aspects that have not been elaborated in two previous studies (Kaldor, 1989; Franco, 1991). Emphasis is given to effects of measurement errors of HPV infection status in biasing the association with cervical neoplasia under more complex scenarios, e.g., differential misclassification between cases and controls in a case-control study, and non-differential misclassification of both viral infection and cervical neoplasia in cross-sectional cytology surveys and cohort studies. Some simple numeric formulae are given that allow the correction of prevalence rates and epidemiological measures of effect, such as the odds ratio and the relative risk, under the latter conditions of misclassification constraint. These formulae have been used to correct estimates from recent epidemiological studies using hypothetical misclassification scenarios in order to obtain clues on the magnitude of the underlying relationship between HPV and cervical cancer.


Interactions between human papillomavirus and human immunodeficiency virus infections. An increasing body of information permits certain conclusions to be drawn about the nature and magnitude of the interactions between HPV and HIV infections and their influence on the genesis of intraepithelial neoplasia and, to a lesser extent, cancer. Importantly, findings tend to be consistent across a number of independent studies. While HPV infection probably does not significantly alter the course of HIV infection, HIV-induced immunosuppression does increase the severity and duration of anogenital warts, increase their infectiousness and reduce treatment efficacy. However, in developed countries the countervailing effects of enhanced HPV infectiousness and declining rates of unsafe sexual behaviour have resulted in stable or declining incidence rates of anogenital warts. Advanced immunosuppression due to HIV infections results in highly significant increases in rates of HPV-associated CIN and AIN. In developed countries, population-based secular trend analyses point to increasing incidence rates of anal cancer in single men in areas of high HIV prevalence, but not yet of cervical cancer in women.


Human papillomaviruses and other biological markers in cervical cancer. The presence of HPV DNA in the genital tract and the genotype of the infecting HPV are now widely employed as biochemical markers in epidemiological studies of cervical cancer. Additional HPV markers could be utilized in future investigations. The amount of HPV DNA is likely to be higher in case specimens than in control specimens; viral genome would be integrated frequently in cases but almost never in controls; and early region transcripts may be relatively more abundant in cases than in controls. When valid serological markers for past HPV infection become available (very likely, antibodies to HPV capsid proteins), they will be useful to estimate lifetime exposure to HPVs. Serological markers for HPV-associated neoplasia (very likely, antibodies to early proteins) may prove useful for surveillance and have prognostic value. A serological marker capable of detecting past herpes simplex virus 2 infection would permit an analysis of the role of this virus in cervical cancer, either as an independent risk factor or in interaction with HPVs. Other possible biomarkers include activation of oncogenes and inactivation of tumour-suppressor genes, assays for serum micronutrients, and analysis of leukocytes for HLA antigens; these should provide insights into the sequence of events that lead to cervical cancer and help to explain the geographic distribution of the disease.


Relationship between human papillomavirus infection and tumours of anogenital sites other than the cervix. Although tissues in the case series of anal, penile, vaginal and vulvar neoplasms that have looked for evidence of HPV infection by probing for HPV DNA have been selected for convenience, they support the view that HPV, especially type 16, is associated with approximately 50% of these tumours. A higher percentage of the anal, vaginal and vulvar tumours are associated with HPV 16 than are penile tumours. This discrepancy may be due to the low number of penile tumours studied or to a true difference in the proportion of penile cancer cases related to HPV. HPV 6/11 and 18 are found less frequently at all anatomic sites. About 10% of tumours that are probed for these viruses are positive, although there are some notable exceptions such as a study that found 39% of penile tumours positive for type 18 and a study that found approximately two thirds of vulvar tumours positive for HPV 18 using Southern blot hybridization. For all of these tumours, there is likely to be a subset of the cases who develop their cancer through mechanisms that do not involve HPV. The case-control studies found a strong association with genital warts, number of sexual partners and, with the exception of vaginal cancer, smoking and/or heavy smoking at the time of diagnosis of the disease. A history of genital warts, smoking at diagnosis, and seropositivity to HSV2 are exposures that have also been found to be associated with cervical cancer. A population-based case-control study in western Washington and Vancouver, British Columbia that studied all anogenital cancers found that a history of genital warts was stronger among patients with vulvar, anal, vaginal and penile cancer than among those with cervical cancer. This was also true of smoking at diagnosis, with the exception of vaginal cancer, where there was little excess risk. This study and other supporting data indicate that these anogenital tumours share many of the same risk factors as cervical cancer.


Comparison studies of HPV detection in areas at different risk for cervical cancer. The hypothesis of a geographical correlation between HPV detection rates and incidence of cervical cancer has been investigated in studies of various types. However, results from these studies are equivocal, in contrast to findings concerning other suspected risk factors which seem to correlate well with the cervical cancer incidence. Possible explanations include (1) greater sensitivity of ecological studies to cumulative exposures such as lifetime number of sexual partners, lifetime smoking and seroprevalence of herpes simplex virus type 2, than to HPV DNA prevalence which does not reflect cumulative exposure to HPV and (2) misclassification in the HPV diagnosis leading to wrong prevalence estimates. In future research, it will be important to establish the sensitivity and specificity of the different methods and conduct intra- and interlaboratory validation studies in order to standardize methods. In spite of the limitations of cross-sectional studies, the measurement of HPV prevalence and its correlation with, for example, sexual behaviour is still valuable for our understanding and interpretation of the role of HPV infections.


Natural history and epidemiological features of genital HPV infection. The spectrum of genital HPV infections comprises clinical, subclinical, and latent disease in addition to HPV-associated neoplasia. The definition of subclinical and latent HPV infection is still incomplete and awaits clarification by highly sensitive HPV detection systems that preserve the morphology of the tissue. Genital HPVs infect the human body mainly by sexual transmission, but other pathways of HPV transmission may be possible as suggested by (a) high prevalences of antibody reactivity in children; (b) lack of association of HPV seropositivity with sexual activity; (c) presence of HPV DNA in oral cavity scrapings of children and adults; and (d) development of recurrent respiratory papillomatosis among children exposed to HPV 6 or 11 during birth. Successful infection depends on the infection site and the immunological state of the host: susceptibility to genital HPVs seems highest for the squamous epithelium of the lower genital tract. HPV DNA is also present in extragenital sites but this is rarely accompanied by clinical or subclinical lesions. The molecular basis for this specific tropism is unknown. The immune response in HPV-infected tissues is characterized by depletion of T helper/inducer cells or Langerhans cells and an impaired immunological function of natural killer cells or the infected keratinocyte. Epidemiological studies indicate that individuals with cell-mediated immunodeficiencies are at increased risk for genital HPV infections. Data about the biological course of genital HPV infections are just beginning to emerge. Regression or persistence of subclinical and latent genital HPV infections as analysed in longitudinal investigations show a constant come-and-go of HPV presence. In an infected individual, complete clearing of the virus seems rather exceptional. With respect to progression, the biological potential of cervical HPV infections is characterized by an increased risk for development of HPV-associated neoplasia, especially in lesions infected with high-risk HPV types (e.g., HPV 16 and 18). Demographic data of genital HPV infections are very variable due to differences in the HPV detection assays used and in the populations examined; the prevalence of subclinical and latent genital HPV infections appears to be at least three times higher compared with clinical HPV infections. This rate increases by a further 3-5-fold when patients are examined several times. Seroreactivity against genital HPV types may be due to an active infection or the result of contact with HPV earlier in life.(ABSTRACT TRUNCATED AT 400 WORDS)


HPV and cervical neoplasia: review of case-control and cohort studies. Epidemiological studies on the association between human papillomavirus (HPV) and cervical cancer are reviewed. Studies using HPV DNA hybridization methods to assess the presence of viral markers have yielded compelling evidence that HPV has a causal role in the disease: the association is strong, consistent and specific to a limited number of viral types. A dose-response relationship has been reported between increasing estimated viral load and risk of cervical cancer. Indirect evidence suggests that HPV DNA detected in cancer cells is a good marker of HPV infection occurring before cancer development. An increased risk for progression to more advanced CIN lesions has been reported among HPV 16/18-positive women as compared to women with other HPV types or to women without any viral DNA.


Screening for cervical cancer: is there a place for incorporating tests for the human papillomavirus? Well organized screening programmes for cervical cancer, based on exfoliative cervical cytology, are known to be effective at reducing the incidence of invasive cervical cancer and mortality from the disease. HPV testing should not replace cervical cytology as the first-line approach in screening for cervical cancer, as HPV testing is not sufficiently reliable and some cancers are not associated with HPV infection. Even though there are many unanswered questions about the validity of HPV tests, it is timely to consider whether HPV testing might improve the management of the substantial number of women whose smears are neither clearly normal nor abnormal, but are described as atypical, suspicious or mildly dyskaryotic. The efficacy and costs of incorporating HPV testing into a cervical cancer screening programme need to be evaluated in controlled trials.


Detection of human papillomavirus in cervical scrapes by the polymerase chain reaction in relation to cytology: possible implications for cervical cancer screening. An HPV screening strategy based on general primer-mediated and type-specific PCR is described. A relationship was found between an increasing HPV prevalence rate and a higher degree of cervical dysplasia (up to 100% in cervical carcinoma). Based on these prevalence studies in the Netherlands and preliminary data showing that progression of cervical lesions is always associated with persistent infection of oncogenic/high-risk HPV types, a cervical cancer screening scheme incorporating PCR-based HPV detection is proposed for use in industrialized countries.


Colposcopic diagnosis of HPV cervical lesions. The colposcopic criteria for abnormal epithelium are leukoplakia, a white area detected after the application of acetic acid or iodine-negative areas not reacting to the acetic acid test. However, white epithelium and iodine-negative areas are not specific for abnormal tissue, condyloma or intraepithelial neoplasia. The lack of specificity of colposcopic findings has motivated the distinction between minor and major grade abnormalities in the recently proposed new colposcopic terminology. Metaplasia and dysplasia cannot be distinguished by colposcopy. All criteria proposed so far lack specificity and reproducibility. Thus, it is at present considered that colposcopy is not a diagnostic method, but an investigative technique that allows the evaluation of the extent of the lesion and localization of the squamo-columnar junction. As for the distinction between condyloma and high-grade intraepithelial neoplasia, all criteria proposed so far also lack reproducibility. Moreover, condyloma is often found at the periphery of high-grade CIN, rendering such a distinction meaningless. The only features specific of HPV infection without dysplasia are condyloma acuminatum and, to a lesser extent, non-acetowhite microcapillary surfaces. Finally, it has been shown that there is no colposcopic sign specific of HPV types.


HPV types in women with normal cervical cytology. PCR-based hybridization methods have been used to show that some women with normal cytology are carriers of HPV DNA of the types strongly related to cervical cancer. How these women should be managed remains unclear. This chapter selectively reviews reports which have estimated type-specific HPV prevalence in relation to the presence or absence of morphological signs of HPV infection. Overall, these reports indicate that among women who were identified as carriers of HPV DNA (by PCR-based methods) and who also had a normal cytological smear, the HPV type detected in the majority of instances was a high-risk viral type for cervical cancer (HPV types 16/18 = 44.7%; HPV types 31/33/35 = 8.1%; other and unknown types = 37.9%). This suggests that screening programmes which include PCR-based HPV detection could reduce the false negative rates currently reported by screening programmes based on cytology alone.


HPV-related genital lesions in men.

HPV-related genital lesions in men. Screening of the asymptomatic male partners of women with genital condyloma or intraepithelial neoplasia has shown that about 50% of the individuals examined present genital HPV-associated lesions. Half to two thirds of these lesions are clinically invisible and are detected only after the acetic acid test. Histological studies have shown that 20% of male partners of women with high-grade intraepithelial neoplasia present lesions histologically defined as high-grade intraepithelial neoplasia. Couples in which both partners present lesions of intraepithelial neoplasia are infected by the same potentially oncogenic HPV type in at least 50% of cases. Also, 50% of subclinical lesions showing only minimal histological changes (acanthosis and papillomatosis, without clear koilocytosis) contain HPV DNA, mostly type 42. In situ hybridization of such lesions indicate HPV-positivity, suggesting that these lesions may be infectious. Cytology does not seem to be specific enough to detect HPV infection in males. Moreover, virological studies do not confirm the hypothesis of an urethral reservoir of HPV. Morphology allows the detection of HPV-associated genital lesions in males. Current treatment protocols allow a 95% cure rate by easily applicable outpatient treatment modalities.


Assessment of exposure to sexually transmitted agents other than human papillomavirus. There are now a number of lines of evidence giving support for a causal role of high-risk oncogenic HPV types in cervical cancer. However, it is clear that only a minority of women with HPV 16 infection, for example, develop invasive cancer. Therefore examination of whether sexually transmitted agents other than HPV are interacting in the genesis of cervical neoplasia is necessary. Serological methods which accurately reflect past exposure to individual agents are ideal for epidemiological studies. Sensitive and specific assays that are available for HSV 1, HSV 2, Treponema pallidum, CMV, HIV and Chlamydia are reviewed. Some data suggest an interaction of herpesviruses with HPV in oncogenesis which future epidemiological studies should address. However, at present the evidence is inadequate to prove the involvement of sexually transmitted agents other than HPV in the etiology of cervical neoplasia.


Angiotensin dependence of endothelium-mediated renal hemodynamics. Endothelium-derived relaxing factor has been shown to regulate renal blood flow, and inhibition of its synthesis increases blood pressure and renal vascular resistance and decreases renal blood flow. Using the substrate antagonist NW-nitro-L-arginine methyl ester (L-NAME), we tested whether renal vasoconstriction induced by endothelium-derived relaxing factor synthesis inhibition could be mediated in part by angiotensin II. In 14 control rats, 10 mg/kg body wt L-NAME increased blood pressure from 106 +/- 6 to 126 +/- 6 mm Hg (p < 0.001), increased renal vascular resistance by 74% (from 19.3 +/- 2.6 to 33.6 +/- 2.9 resistance units), and decreased renal blood flow by 34% (from 5.9 +/- 0.5 to 3.9 +/- 0.3 ml.min-1.g kidney wt-1, p < 0.005). When six rats were treated with 10 mg/kg body wt of the angiotensin receptor antagonist DuP 753, L-NAME increased blood pressure from 84 +/- 4 to 106 +/- 4 mm Hg (p < 0.001); however, renal vascular resistance increased by only 27% (from 13 +/- 2 to 17 +/- 3 resistance units, p < 0.01; p < 0.05 different from control value) and renal blood flow was unchanged. Likewise, after pretreatment of six rats with 32 micrograms/100 g body wt of the angiotensin converting enzyme inhibitor enalaprilat, L-NAME increased blood pressure from 88 +/- 5 to 124 +/- 6 mm Hg (p < 0.001) and renal vascular resistance by 54% (from 12 +/- 1 to 18 +/- 3 resistance units, p < 0.01; p < 0.05 different from control value) but renal blood flow was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)


Arachidonic acid- and acetylcholine-induced relaxations of rabbit aorta. The present study investigated the role of arachidonic acid and acetylcholine in mediating endothelium-dependent relaxations of rabbit aorta. Isolated thoracic aortic rings were precontracted with a submaximal concentration of norepinephrine, and the effect of various agents on arachidonic acid- and acetylcholine-induced relaxations was examined. Arachidonic acid elicited a concentration-related relaxation that was potentiated by the cyclooxygenase inhibitor indomethacin. Treatment with the lipoxygenase inhibitor nordihydroguaiaretic acid completely blocked but the cytochrome P450 inhibitor metyrapone had no effect on arachidonic acid-induced relaxation. NG-Monomethyl-L-arginine and nitro-L-arginine, compounds that inhibit the nitric oxide-like endothelium-derived relaxing factor, had little or no effect on arachidonic acid-induced relaxations. In contrast, nordihydroguaiaretic acid, metyrapone, NG-monomethyl-L-arginine, and nitro-L-arginine all attenuated the relaxation to acetylcholine; however, indomethacin had no effect on acetylcholine-induced relaxations. Arachidonic acid and acetylcholine had no effect on denuded rabbit aorta. Incubation of rabbit aorta with [14C]arachidonic acid resulted in the synthesis of major radioactive metabolites that comigrated with the prostaglandins and hydroxyeicosatetraenoic acids. Indomethacin selectively inhibited prostaglandin formation, nordihydroguaiaretic acid attenuated both prostaglandins and hydroxyeicosatetraenoic acids, and metyrapone blocked the epoxyeicosatrienoic acids. Additionally, acetylcholine elicited a twofold increase in tissue cyclic guanosine monophosphate content in contrast to a 59% reduction in cyclic guanosine monophosphate content observed with arachidonic acid. Therefore, these data suggest that in rabbit aorta, arachidonic acid-induced relaxations are mediated by an endothelium-dependent factor (or factors) that differs from the factor (or factors) released by acetylcholine. These results support the existence of multiple endothelium-derived relaxing factors.


Platelet activating factor amplifies human neutrophil adherence to bovine endothelial cells: evidence for a lipoxygenase dependent mechanism. Platelet activating factor (PAF) is a potent lipid mediator that induces the release of leukotrienes and prostaglandins from various cells and tissues. We examined the capacity of PAF alone and in combination with soluble stimuli to enhance eicosanoid synthesis and adherence of human neutrophils. Neutrophils were preincubated with PAF and washed before exposure to the soluble stimuli F-Met-Leu-Phe (FMLP), calcium ionophore A23187, and phorbol myristate acetate. Preincubation of neutrophils with 1 microM PAF enhanced the release of both LTB4 and LTC4 in response to each of the three agonists, in contrast with the unprimed neutrophils. Priming was specific for PAF since lyso-PAF was inactive. Priming concentrations of PAF also augmented the adherence of neutrophils to endothelium in the presence of the soluble agonists A23187, phorbol myristate acetate, and FMLP. The priming effect of PAF on eicosanoid release and neutrophil adherence was shown to have similar time- and dose-dependent effects. Further, the priming effects of PAF on adherence could be reversed by preincubation of neutrophils with the lipoxygenase inhibitors nordihydroguiaretic acid and 5,8,11,14-ETYA but not by preincubation with the cyclooxygenase inhibitor indomethacin. These data demonstrate that PAF amplifies neutrophil adherence to endothelium through a lipoxygenase dependent mechanism.


Time course of superoxide generation by leukocytes--the MCLA chemiluminescence system. This study was performed to examine the pattern of superoxide (O2-.) generation from leukocytes using the O2-. specific chemiluminescence (CL) method. Cypridina luciferin analog, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo [1,2-alpha]pyrazin-3-one (MCLA) was used as a CL probe. The appropriate conditions of the MCLA method was first determined for the evaluation of the time course of O2-. generation by leukocytes. The time course of O2-. generation obtained by the MCLA-CL system was compared with that by the luminol-dependent CL, electron spin resonance (ESR)/spin trapping, and cytochrome c systems. Following stimulation by three different stimulants (PMA, OZ, FMLP), leukocytes continuously generated O2-. for up to 5 h in the MCLA-CL system, irrespective of the kind of stimulation. The curves obtained by generation ceased more rapidly in the luminol-CL, ESR/spin trapping, and cytochrome c systems. A 50% activity of the initial value was observed at 70 min in the MCLA-CL system, but 30, 10 and 35 min in the other systems, respectively. The CL or O2-. generation value decreased to less than 1% (possible termination) at 300, 90, 120 and 180 min, respectively. With the exception of ESR studies with OZ, the cell viability was not significantly affected in any of the trials. These results indicate that leukocytes can generate O2-. much longer than previously estimated and that the MCLA-CL-system is the most suitable system for the measurement of the O2-. generation by leukocytes.


Granulocyte turnover in the feline intestine. The objective of this study was to determine the turnover rate of the extravascular pool of granulocytes in different regions of the feline gastrointestinal tract. Leukocyte emigration from the vasculature was prevented over a 48-h period by repeated intravenous injections of a monoclonal antibody (MAb IB4) directed against the leukocyte adhesion glycoprotein complex CD11/CD18. Tissue-associated myeloperoxidase (MPO) activity was used to monitor the total tissue granulocyte pool at 0.5, 12, 24, and 48 h after MAb IB4 administration. The mucosal layer of the duodenum, jejunum, ileum, and colon exhibited different kinetics of granulocyte clearance, with average life-spans (t1/2) ranging between 6.9 (colon) and 10.4 h (duodenum). Granulocyte clearance rates of 0.5 x 10(6) and 2.4 x 10(6) cells/h/g tissue were estimated (from measured values of t1/2 and tissue granulocyte pool) for the small bowel and colonic mucosae, respectively. The submucosal layer of the intestine exhibited a biphasic reduction in tissue MPO activity following immunoneutralization of CD11/CD18, with an initial t1/2 < or = 0.5 h followed by a t1/2 of 36-60 h. The initial rapid decline in tissue MPO suggests that a significant fraction of granulocytes in the submucosa is localized in a readily exchangeable pool (e.g., marginated cells within the vasculature). The results of this study indicate that the average life-span of resident granulocytes varies significantly between different regions of the gastrointestinal tract, with the intestinal mucosa exhibiting a t1/2 comparable to that previously reported for circulating feline neutrophils (approximately 8 h).


Epidemiology of Japanese encephalitis.

Epidemiology of Japanese encephalitis. Seven hundred and sixty-two cases of Japanese Encephalitis (JE) were studied during the last 5 years (1985-1989) in relation to age, sex, religion, nutritional status, living habits, exposure to domestic animals and mosquitos, clinical profile, seasonal variation and mortality pattern. The maximum occurrence was in 1987-1988 and it showed a preponderance in males (51-82%). The disease is progressively decreasing in Muslims (3-7%) and gradually increasing in tribes (25-60%). Children in the age-group of 6-7 years (19-25%) were maximally affected and the disease was rare in infancy. The common features were coma, convulsions, neck rigidity and fever (88-97%). Gastrointestinal manifestations were rare (3.6%) but were associated with the highest mortality. About 80-95% had exposure to domestic animals directly or indirectly and 95% of the patients were not using mosquito nets. The CSF protein and sugar content were normal, with or without slight leucocytosis while the lymphocyte count was variable. The CSF and blood picture had no significant relation with clinical presentation and prognosis.


Expression of carcinoembryonic antigen and related genes in lung and gastrointestinal cancers. Carcinoembryonic antigen (CEA), a tumor marker for lung cancers of small cell (SCLC) and non-small cell (NSCLC) types, belongs in a multigene family which includes non-specific cross-reacting antigen (NCA) and biliary glycoprotein 1 (BGP). We used specific cDNA probes and a CEA immunoassay to determine the pattern of expression in normal and malignant lung and gastrointestinal (GI) tissues. Normal lung contained high amounts of NCA and a low concentration of CEA. All 3 genes were expressed discordantly in lung tumors and cell lines. In contrast, all three genes were expressed in most G1 tumor cell lines. In both lung and colorectal cell lines expression of NCA RNA was relatively high, while BGP RNA was relatively low, and the median concentrations of CEA were greater than in corresponding non-malignant tissues. While CEA protein concentrations in lung cell lines were similar to those present in G1 cell lines, the ratio of NCA:CEA RNA was significantly higher in lung cancer lines than in colorectal lines. Thus, NCA constitutes most of the "CEA-like" immunoreactivity previously described in lung cancers. There was excellent concordance between expression of CEA RNA and CEA protein, as well as between concentrations of CEA protein in cell line pellets and supernatant fluids. Of interest, significantly higher rates of CEA expression were present in lung cancers expressing neuroendocrine (NE) markers. The association between CEA expression and NE cell properties is intriguing and may prove to be of clinical interest.


Correlation between the serum level of hepatitis C virus RNA and disease activities in acute and chronic hepatitis C. The influence of viremia on hepatic injury in patients infected with hepatitis C virus was examined by analysis of the relationship between alanine aminotransferase activity and the amount of hepatitis C virus RNA in sequential serum samples from I untreated patient with acute hepatitis C and 3 untreated patients with chronic hepatitis C. Semiquantitative analysis by the competitive-reverse-transcription/polymerase-chain-reaction method indicated that the quantity of hepatitis C virus RNA in the serum affected the disease activities of acute and chronic hepatitis C through their natural clinical courses in all these patients. The nucleotide sequence encoding the putative envelope region of the viral genome in the patient with acute hepatitis C was examined. Blood samples taken serially at 2 times of exacerbation of the hepatitis revealed 2 nucleotide mutations, resulting in changes of predicted amino acid residues. This finding suggests that nucleotide mutations in the envelope region of the viral genome may be responsible for the recurrent hepatic injury attributed to recurrence of viremia in patients with hepatitis C. From these aspects, the serial divergence of the virus genome in infected individuals, especially in the region encoding the viral envelope protein, may possibly play an important role in developing chronic infection of hepatitis C virus.


Viral co-infections in human papillomavirus-associated anogenital lesions according to the serostatus for the human immunodeficiency virus. In HIV-infected men, human papillomavirus (HPV) infection is strongly linked with the development of anogenital lesions but is not a sufficient factor to explain the neoplastic transformation of such lesions. We investigated the association between HPV and herpesvirus infections in penile and anal lesions from 54 HIV-seronegative and 54 HIV-seropositive men by means of colposcopy, histopathology and in situ hybridization. Our patients showed condyloma acuminata (39%), papular warts (35%) and macular warts (26%). High-grade lesions were predominant in the HIV+ men, whereas low-grade lesions were more frequent in the HIV- men. In the HIV+ group, potential oncogenic HPV were the most frequently detected (83.4%) whereas the "low-risk" HPV were found chiefly in HIV- men (62.1%). The CD4 number was lower in patients showing "high-risk" HPV than in men showing lesions without HPV or with non-oncogenic HPV. HPV types 6/11 were found mainly associated with koilocytosis or with AIN(PIN)I. Oncogenic HPV were more often detected in AIN(PIN)II-III. The herpesviruses DNA detection revealed a higher prevalence of HSVI and -2 than CMV and EBV in the studied biopsies. The frequency of HSV and CMV detection was higher in the HIV+ than in the HIV- men. A link was found between the "high-risk" HPV and the CMV detection whatever the population considered. The detection in HPV lesions of other sexually transmitted viral agents could therefore represent an important means of preventing progression of the anogenital disease, especially in immunosuppressed patients.


Interaction of gangliosides with the methyltransferase-I of microsomes from normal and neoplastic human mammary gland. The activity of the phospholipid methyltransferase-I, which catalyzes the rapid transmethylation of phosphatidylethanolamine to phosphatidylcholine, was found to be about 6-fold enhanced in microsomal membranes of breast cancer with respect to the level found in normal human mammary gland. Exogenous gangliosides GM1 and GM2 added to neoplastic breast microsomes induced progressive inhibition of the methyltransferase activity. In contrast, in microsomal membranes of non-neoplastic breast tissue treated with these gangliosides, the methyltransferase activity was markedly increased. The addition of cholesterol to these microsomes led to complete inhibition of the GM1-stimulated enzyme activity. The methyltransferase activity was not affected by GM3 alone in either type of tissue. Experiments carried out on non-neoplastic microsomes revealed that the phospholipid methyltransferase-I was affected by that portion of gangliosides which remained stable associated to microsomal membranes.


The causal link between human papillomavirus and invasive cervical cancer: a population-based case-control study in Colombia and Spain. To evaluate the association between human papillomavirus (HPV) and cervical cancer, we performed a population-based case-control study in Columbia and Spain, the former country having an incidence rate of cervical cancer about 8 times higher than the latter. It included 436 cases of histologically confirmed invasive cervical cancer and 387 randomly selected population controls. Information on demographic variables, sexual behaviour and other risk factors was obtained by interview. HPV-DNA was measured in cervical-swab specimens with 3 hybridization assays: ViraPap, Southern hybridization (SH) and polymerase chain reaction (PCR). The presence of HPV-DNA and detection of types 16, 18, 31, 33 and 35 were strongly associated with cervical cancer in each country regardless of the assay used. For both countries combined the adjusted odds ratios and 95% confidence intervals were: ViraPap OR = 25.9 (10.0-66.7); SH OR = 6.8 (3.4-13.4); and PCR OR = 28.8 (15.7-52.6). HPV-16 was the most common type detected in both cases and controls. Our results indicate that there is a very strong association between HPV 16, 18, 31, 33 and 35 and invasive cervical cancer and that this association is probably causal.


Risk factors for cervical cancer in Colombia and Spain. A population-based case-control study of cervical cancer was conducted in Spain and Colombia to assess the relationship between cervical cancer and exposure to human papillomavirus (HPV), selected aspects of sexual and reproductive behaviour, use of oral contraceptives, screening practices and smoking. The study included 436 cases of histologically confirmed squamous-cell carcinoma and 387 age-stratified controls randomly selected from the general population that generated the cases. The presence of HPV DNA in cervical scrapes was assessed by PCR-based methods and was the strongest risk factor (OR = 23.8; 13.4-42.0). Risk estimates for any other factor were only slightly modified after adjusting for HPV status. Among women found positive for HPV DNA, only the use of oral contraceptives was a risk factor for cervical cancer (OR = 6.5; 1.3-31.4 for ever vs. never use). Patients with cervical cancer who were HPV DNA-negative retained most of the established epidemiological features of this disease. This suggests that some instances of HPV infection went undetected or that other sexually transmitted factor(s) contribute to the causation of cervical cancer. Early age at first intercourse (OR = 4.3; 2.1-9.0 for age < 16 vs. 24+) and early age at first birth (OR = 5.0; 1.8-14.2 for age < 16 vs. 24+) were associated with increased risk of cervical cancer; these effects were independent of one another. Low educational level was a risk factor (OR = 2.5; 1.6-3.9). Number of sexual partners was in our study a surrogate for HPV infection. Smoking and parity after age 24 were weakly and inconsistently associated with the risk of cervical cancer. Previous screening (OR = 0.7; 0.5-1.0) and ever having undergone a Caesarean section (OR = 0.4; 0.2-0.8) were protective factors.


Transforming growth factor beta 1 (TGF-beta 1) inhibits growth of a human ovarian carcinoma cell line (OVCCR1) and is expressed in human ovarian tumors. The effects of EGF and TGF-beta 1 on the proliferation of 2 ovarian carcinoma cell lines (IGROV1 and OVCCR1) were evaluated. The cell lines were adapted to grow in a restricted serum (0.5%) medium. EGF was required for proliferation of both ovarian cell lines. Low doses of TGF-beta 1 inhibited clonogenic capacity and attenuated the EGF-mediated stimulation of DNA synthesis in OVCCR1 cells. TGF-beta 1 inhibited OVCCR1 cell proliferation by blocking the cell cycle at the G1/S transition. TGF-beta 1 did not affect either clonal or monolayer growth of IGROV1 cells. Both cell lines express type-I and type-III TGF-beta receptors, suggesting that the unresponsiveness of IGROV1 cells to TGF-beta 1 occurs at a post-receptor level. TGF-beta 1 mRNA was detected in OVCCR1 cells and in 8 out of 11 of the ovarian tumor specimens examined.


Early superoxide dismutase alterations during SV40-transformation of human fibroblasts. The expression of superoxide dismutases (SOD) 1 and 2 was studied in 4 clones of human fibroblasts after their infection by simian virus 40 (SV40), in parallel with the alterations of chromosomes 21 and chromosome 6q arms, carrying the genes that encode for SOD1 and SOD2 respectively. For all clones, a similar scheme with 2 main phases was observed for both chromosome and SOD variations. The first phase, defined as the pre-crisis phase, was characterized by chromosomal instability, but maintenance of normal numbers of chromosome 6q arms and chromosomes 21. The level of SOD2 mRNA was high, while SOD2 activity and immunoreactive protein were low. SOD1 protein and activity were decreased. In the second phase, defined as the post-crisis phase, the accumulation of clonal chromosomal rearrangements led to the loss of 6q arms, while the number of chromosomes 21 remained normal. SOD2 mRNA level was decreased and SOD2 immunoreactive protein and activity remained low. SOD1 protein and activity increased with passages, reaching values similar to those of control cells at late passages. As in established SV40-transformed human fibroblast cell lines, good correlation was found between SOD2 activity and the relative number of 6q arms. These results allow us to reconstruct the sequence of events leading to the decrease of SOD2, a possible tumor-suppressor gene, during the process of SV40-transformation of human fibroblasts.


Expression and function of VLA-alpha 2, -alpha 3, -alpha 5 and -alpha 6-integrin receptors in pancreatic carcinoma. The expression of the VLA-integrins alpha 2, alpha 3, alpha 5 and alpha 6 was studied immunohistochemically in tissue samples from ductal pancreatic cancer, chronic pancreatitis, normal pancreas and in 8 cell lines of ductal human pancreatic cancer. Furthermore, adhesion assays on purified extracellular matrix (ECM)-compounds were used to define the function of alpha 2, alpha 3, alpha 5 and alpha 6 in pancreatic cancer cells. Immunohistochemically, VLA alpha 2 and VLA alpha 6 were moderately to strongly expressed on the basal surface of ductal and acinar cells in normal pancreatic tissue, while centro-acinar cells predominantly expressed VLA alpha 3 and VLA alpha 5. Pancreatic carcinoma showed intense staining for VLA alpha 2 and VLA alpha 6 with a diffuse distribution on the cell surface. The redistribution of VLA alpha 2 and VLA alpha 6 may reflect a loss of spatial arrangement of tumor cells and their ability to interact randomly with extracellular matrix structures during invasion and metastasis. Expression of VLA alpha 3 and VLA alpha 5 in pancreatic carcinoma was heterogeneous, ranging from moderate to weak, and was lost in about 50% of the cells. Two pancreatic carcinoma cell lines (PC 3, PC 44) were further investigated in adhesion assays. Monoclonal antibodies (MAbs) against alpha 2 (GI 9, 10-G-11) were able to inhibit tumor-cell adhesion to collagen IV (59%-72%) in both cell lines. A MAb against alpha 6 (GoH3) inhibited tumor-cell adhesion to laminin (52%-86%) in both cell lines. These results suggest that alpha 2 is a collagen-binding site and alpha 6 a laminin-binding site in pancreatic cancer cells. The anti-alpha 5-MAb SAM I inhibited adhesion of PC3 to fibronectin (76%), being without effect in PC44. Adhesion of both cell lines to fibronectin was almost completely inhibited by RGDS (85%-88%). Thus, alpha 5 is a functionally important fibronectin binding site in some pancreatic carcinoma cells, suggesting further RGD-dependent fibronectin binding sites in other pancreatic carcinoma cells.


Decreased expression of E-cadherin and increased invasive capacity in EBV-LMP-transfected human epithelial and murine adenocarcinoma cells. The EBV-encoded membrane protein LMP is one of 9 viral proteins regularly expressed in virally immortalized B lymphocytes. It is expressed in EBV-carrying lymphoblastoid cell lines of normal origin and in the majority of the poorly differentiated nasopharyngeal carcinomas, but not in Burkitt lymphomas. LMP has been reported to transform rodent fibroblasts, to inhibit epithelial differentiation and to alter morphology and cytokeratin expression in an in vitro immortalized human keratinocyte cell-line, RHEK-I. We now report that an LMP-transfected mouse mammary carcinoma line, SHG, exhibits a similar morphological change to that previously described in the LMP-transfected RHEK-I. In the LMP-transfected RHEK-I and SHG cells, we observed a decreased expression of the calcium-dependent adhesion molecule E-cadherin. The LMP-transfected RHEK-I cells were capable of invading type-I collagen gels while the control cells were not. The LMP-transfected SHG cells showed a significantly higher invasive capacity than the original cell line.


Cardiac beta-adrenergic mediated chrono- and inotropic effects of imipramine in vitro. Clinical and experimental studies show that tricyclic antidepressants in "therapeutic plasma concentrations" can increase heart rate, myocardial contractility and blood pressure. Our study was undertaken to analyze the role of beta-adrenergic stimulation in the chronotropic and inotropic effects of imipramine. Strips of rat right atrium including the sinus node, which were beating spontaneously, were used to study chronotropism. Strips of the left atrium, electrically stimulated to beat at 1 Hz, were used to study inotropism. The preparations were superfused in vitro with Tyrode's solution at 37 degrees C and exposed to imipramine while recording membrane potentials or force of contraction. Imipramine exerted dose-dependent biphasic actions. Imipramine 0.8 microM produced positive chronotropic and inotropic actions which were blocked by propranolol. Imipramine 1.6 microM depressed the sinus node automaticity, but it did not modify the force of contraction. Imipramine 3.2 microM depressed both the sinus node automaticity and the myocardial contractility. In conclusion, imipramine in "therapeutic plasma concentrations" produces beta-adrenergic mediated cardiac positive chronotropic and inotropic actions. The possible mechanisms of the depressant effects of imipramine itself on automaticity and contractility are still not clear. The results presented can explain stimulatory and depressant cardiac effects of therapeutic doses and overdoses of tricyclic antidepressants.


Increased expression of atrial myosin light chain 1 in the overloaded human left ventricle: possible expression of fetal type myocytes. We examined the isoforms of myosin light chain 1 in the human left ventricles using pyrophosphate and sodium dodecyl sulfate polyacrylamide gel electrophoresis, peptide mapping, and immunoblotting with monoclonal antibodies against human atrial light chain 1. The relationship between hemodynamic parameters and light chain 1 isoform composition was compared among groups of patients with hypertrophic cardiomyopathy (n = 8), dilated cardiomyopathy (n = 9) and aortic stenosis (n = 5), and controls (n = 6). (1) The light chain 1, which differed from ventricular light chain 1 found in the normal adult ventricle, was highly expressed in the overload left ventricle, and was identical to atrial and fetal ventricular light chain 1 with respect to the physiochemical and immunological properties. (2) The expression of atrial/fetal light chain 1 was augmented in the subendocardial area in comparison with the mid- or subepicardial areas in the hypertrophied left ventricles. (3) The values (%) of the relative expression of atrial/fetal light chain 1 to total light chains 1 determined by densitometric analysis were significantly higher in patients with dilated cardiomyopathy (40.2 +/- 5.8) and those with aortic stenosis (43.1 +/- 6.2) than in the controls (16.9 +/- 2.5) (p less than 0.01), but there was no significant difference between the patients with hypertrophic cardiomyopathy (28.0 +/- 3.7) and the controls. (4) The values of the ratio significantly correlated with those of peak circumferential wall stress (r = 0.53, p less than 0.005). These results suggest that atrial/fetal light chain 1 is expressed in the left ventricles in response to the increased hemodynamic load.


Specific and high affinity binding of perindoprilat, but not of perindopril to blood ACE. The bindings of perindopril and of its active metabolite perindoprilat to human serum, isolated proteins and to erythrocytes were studied by equilibrium dialysis. Within the therapeutic concentrations range, perindopril was 74% bound to serum involving a non-saturable process, NKa = 2.87. The main binders are serum albumin and alpha 1-acid glycoprotein. The serum binding of perindoprilat involved two successive steps. First, a saturable high-affinity binding (Ka: 2.8 x 10(9) M-1) occurred, involving probably the angiotensin converting enzyme (ACE). The second binding step was non-saturable with a very weak binding capacity, NKa = 0.15, quite superimposable to the HSA bound perindoprilat. Free fatty acids (FFA) did not alter the binding to HSA. The binding of both compounds to erythrocytes was low especially with perindopril, when measured in the presence of plasma. A significant correlation showed that the overall serum binding percentage of both drugs was essentially determined by HSA concentration. Serum binding was decreased in renal failure or cirrhosis, this result was principally linked to the hypoalbuminemia. Interactions with other drugs were limited to the binding of salicylate, tolbutamide and digitoxin to HSA.


Placebo effect in cardiovascular clinical pharmacology. A placebo is a pharmacologically inactive substance that can have a therapeutic effect if administered to a patient who believes that he or she is receiving an effective treatment. It is generally admitted that the placebo effect decreases blood pressure in 20% to 30%, when evaluated by casual sphygmomanometer or ambulatory systems. In order to evaluate the occurrence of a placebo effect in cardiovascular pharmacology, we analysed two studies. One study included ten mild-to-moderate hypertensive patients, and consisted of two submaximal exercise tests separated by a single oral administration of the placebo. The other study included six healthy volunteers, receiving an oral placebo during ten days. The placebo was in both case presented as an effective antihypertensive agent. Any change on blood pressure and heart rate, both at rest and during exercise, was observed before and three hours after the placebo. After ten days of placebo administration, no statistically significant change in blood pressure or heart rate was obtained. Nor was any statistical difference observed in catecholamine plasma levels, either three hours or ten days after oral administration of the placebo. The second study did not show any evidence of changes in lymphocytic beta-adrenoceptor density after ten days of placebo. The results of these pilot studies suggest that the use of a placebo group in cardiovascular clinical pharmacology should be reconsidered. The real occurrence and characteristics of a placebo effect should be evaluated by a complementary study.


Host resistance assays as predictive models in styrene immunomodulation. Three infection models namely an oncogenic virus Encephalomyocarditis (EMCV), a rodent strain of malaria, Plasmodium berghei, and a rodent hookworm parasite, Nippostrongylus brasiliensis, were used to confirm the in vivo immunotoxic potential of styrene reported in our previous communication. The altered host resistance to these challenge infections was evaluated in rodents pre-treated with 0, 0.02, 0.03 or 0.05 x LD50 dose of styrene (5 days/week) for 4 weeks. Significantly increased mortality in mice was observed at the various tested dose levels of styrene when challenged with EMCV. Similarly the results obtained in the malaria infection model indicated increased blood parasitaemia as well as significantly enhanced mortality in styrene-treated animals. Also the rejection of N. brasiliensis was also found to be significantly impaired in animals treated with a higher dose of styrene. These results indicate that the exposure of rodents to styrene can markedly impair host resistance which may have biological significance.


Lack of immunosuppression by ketoconazole and itraconazole. The antifungal drugs ketoconazole and itraconazole were evaluated for their effects in the following test systems: in vitro, phytohaemagglutinin (PHA)-induced proliferation of human peripheral blood mononuclear cells and IL-2-driven proliferation of CTLL-2 cells; in vivo, antibody response to sheep red blood cells (SRBC) and delayed-type hypersensitivity (DTH) reaction to oxazolone. At a concentration of 10 microM, ketoconazole moderately and itraconazole strongly inhibited thymidine (Thd) incorporation in human peripheral blood mononuclear cells cultured in medium supplemented with 5% human serum. Increasing the serum concentration from 5 to 20% almost completely reversed these inhibitory effects. Also, cell viability, found to be less than 15% in cultures containing 10 microM itraconazole was restored by increasing the serum concentrations in the culture medium. Similar observations were made in experiments using IL-2-stimulated CTLL-2 cells: the growth inhibition in the presence of 10 microM ketoconazole or 1 microM itraconazole could be counteracted by increased serum supplementation. In vivo, subchronic intraperitoneal dosing with 40 mg/kg ketoconazole or itraconazole to mice had no effect on the antibody response to SRBC as measured by the number of splenic IgM and IgG plaque-forming cells and did not significantly affect the DTH response to oxazolone. These data indicate that neither ketoconazole nor itraconazole exert immunosuppressive properties in vivo. Their in vitro inhibitory effects on PHA-induced lymphocyte proliferation and IL-2-dependent CTLL-2 growth are reversed by the serum supplementation to the culture medium and these activities should therefore be considered as in vitro artefacts.


Differing effects of delta-9-tetrahydrocannabinol (THC) on murine spleen cell populations dependent upon stimulators. Delta-9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, can suppress the immune response, both in vitro and in vivo. In the present study, THC was found to either up-regulate or down-regulate lymphocytes depending on the method of stimulation. When the mitogens concanavalin A (Con A) or phytohemagglutinin (PHA) were used to stimulate THC-treated splenocytes, a down-regulation of lymphocyte proliferation occurred, which reflected lower T-cell numbers in general and Ly2 positive cells specifically. When splenocytes were stimulated directly by using anti-CD3 antibody it was found that low concentrations of THC enhanced lymphocyte proliferation, T-cell numbers in general, and Ly2 cells specifically. These results emphasize that THC can either enhance or suppress aspects of the immune response, depending on the specific immune stimulants used and the specific parameter of immunity measured.


Plasma levels of polymorphonuclear elastase and myeloperoxidase after uphill walking and downhill running at similar energy cost. This study was designed to compare the effects of eccentric and concentric exercises on blood polymorphonuclear neutrophil count (nPMN) and plasma levels of myeloperoxidase ([MPO]) and elastase ([EL]) used as markers of neutrophil (PMN) activation. Ten healthy male subjects underwent 2 periods of exercise of 20 min duration each at 60% VO2max on an inclined treadmill. They initially walked up a 5% grade (UW). Ten days later they ran downhill on a 20% grade (RD). Blood samples were collected 10 min before and immediately after exercise. A postexercise sample was drawn after 20 min recovery. Both exercise bouts were accompanied by a similar and significant (p < 0.01) increase of 33% in nPMN above resting values. Baseline nPMN values were reached after 20 min recovery. There were no significant changes in [MPO] and [EL] following UW. In contrast, RD was followed by significant increases (p < 0.001) in [MPO] (+97%) and [EL] (+70%) above resting levels. While [MPO] returned to its pre-exercise level after 20 min recovery, [EL] remained significantly elevated (p < 0.05). These results clearly demonstrate the importance of the eccentric component of muscle contraction in exercise-induced PMN activation.


CTLA-4 and CD28: similar proteins, neighbouring genes. Subtractive cloning and screening yielded a cDNA clone corresponding to a molecule expressed in activated T cells, called CTLA-4. At the protein level, CTLA-4, a single-V-domain member of the immunoglobulin superfamily, was found very homologous to the lymphocyte activation molecule CD28. In particular, the hinge region included the hexamer MYPPPY, completely conserved for both molecules and in mice and humans. By immunizing mice with a human CTLA-4 peptide, an anti-CTLA-4 monoclonal antibody (MAb) was obtained, which enabled to establish the MW of the protein (26 and 40 kDa under reduced and non-reduced conditions respectively) and its preliminary tissue distribution. Also, CTLA-4 and CD28 were very similar at the message and at the gene structure level. The corresponding genes had previously been found to co-map on mouse chromosome IC and on human chromosome 2q33. We show that they can be found on the same yeast artificial chromosomes bearing human genomic DNA, and that they are 25 to 150 kb apart. These marked homologies and gene proximity strongly suggest that CTLA-4 and CD28 are the direct products of a duplication event, and raise the question of the function of CTLA-4.


CD28 co-stimulation of T-cell-mediated cytotoxicity. Co-stimulation via the CD28 pathway permits small, resting human peripheral-blood T lymphocytes to mediate anti-CD3 monoclonal antibody (MAb) "re-directed" cytotoxicity. The effector cells are contained with the "memory" population of T lymphocytes, identified by expression of the CD45RO differentiation antigen. In this article, we review the requirements for initiating a cytolytic response and speculate on the physiological consequences of this process.


Itraconazole in the treatment of chromoblastomycosis due to Fonsecaea pedrosoi. The efficacy and tolerability of itraconazole in chromoblastomycosis due to Fonsecaea pedrosoi were evaluated in a non-comparative open clinical trial in 19 Brazilian patients with histopathologically and mycologically proven active chromoblastomycosis. Patients were classified in terms of severity and received itraconazole at the dosage of 200 to 400 mg per day until previously described criteria of cure have been reached. Clinical, mycologic, histopathologic, and laboratory evaluations were performed before, during, and after therapy. The plasma levels of itraconazole and the in vitro susceptibility of the isolates were determined in 15 cases. Clinical and biologic cure were achieved by eight patients (42%) having mild to moderate disease, after a mean duration of therapy of 7.2 months (3.2-29.6 months). Sterile scarred lesions were observed in a post-therapy follow-up lasting on average 9.6 months that was carried out in this subgroup. Clinical cure alone occurred after a mean period of 25.1 months of treatment (16-30.5 months) in seven patients (36%) with moderate to severe disease. Finally, clinical improvement was obtained in four patients (21%) with severe lesions after a mean treatment time of 17.6 months (10.7-22.5 months). All patients responded favorably to itraconazole therapy. No significant side effects nor biochemical alteration during this trial were important enough to interrupt the treatment. Our results support those of previous trials, suggesting that itraconazole is an effective compound against chromoblastomycosis due to Fonsecaea pedrosoi.


Uniformity of serotype and electropherotype in local human rotavirus isolates during each of three successive cold seasons. Each of three consecutive cold seasons (November-March) in the town of Tiberias, Israel, was dominated by one particular rotavirus serotype causing acute diarrhoea in the community: the 1987/88 season by serotype-2; 1988/89 by serotype-1 and 1989/90 by serotype-4. Each season was also characterized by a particular pattern of rotaviral RNA when visualized using electrophoresis in gels. RNA profiles of identical rota serotypes and serotypic prevalence for any given cold season were unique for the town of Tiberias and different from other localities throughout Israel. The meaning of these findings in terms of herd immunity is discussed.


Risk factors of obesity in a five year old population. Parental versus environmental factors. To be more effective, the prevention of obesity in childhood should be focused on the population at risk. The purpose of the present study is firstly to find correlations between certain environmental factors and obesity in childhood, and secondly to measure the influence of the environmental factors after taking the parental history of obesity into account. This case controlled study includes 704 controls vs. 327 cases selected in a population of five year old school children. The anthropometric assessment was completed at school. Obesity was defined as a weight for height > or = 2 s.d. using the French weight charts for French children based on sex and height. Interviews of the parents recorded parental overweight and child birth overweight as 'constitutional' factors and family structure, socio-economic level and daily lifestyle (sleep, TV viewing, after school care, etc.) as 'environmental' factors. The results show that parental overweight and birth overweight are closely related to the child's obesity at five years of age (estimated relative risks 3.1 and 2.4 respectively). The environmental factors which contribute to child obesity are: southern European origin of the mother, snacks, excessive television viewing and, more importantly, short sleep duration (estimated relative risks = 1.9, 1.3, 2.1 and 4.9 respectively). A logistic regression model, after taking parental overweight into account, shows that the relationship between obesity and short sleep duration persists independently of television viewing. The hypotheses raised by these findings are discussed.


Assessment of abdominal fat distribution in obese patients: anthropometry versus computerized tomography. The accuracy of waist to hip girth ratio (WHR) in assessing visceral/subcutaneous abdominal fat distribution has not yet been clearly established in the obese population. The purpose of the present study was to evaluate the relationship between WHR and visceral/subcutaneous fat distribution, both assessed by computerized tomography (CT), in a group of 28 obese patients (15 male, 13 female). Furthermore, 33 normal weight or slightly overweight subjects (23 male, 10 female) were studied as a control group. Obese subjects of both sexes were found to have higher values of WHR than non-obese; conversely visceral:subcutaneous fat area ratio (VSR) values did not differ significantly. Significant correlation between WHR and BMI was present both in males (r = 0.41, P < 0.01) and in females (r = 0.54, P < 0.01). In normal weight males significant correlations between WHR and visceral fat area or VSR were found. In obese males these correlations were much weaker. In normal weight females a significant correlation was found between waist circumference and visceral fat area, whereas in obese females no positive correlations were found between anthropometric measurements and CT indices of visceral fat distribution. In conclusion, WHR cannot be considered as a reliable index of visceral/subcutaneous fat distribution in obese patients, particularly if they are females.


Adipose cell size and distribution in familial lipoprotein lipase deficiency. To determine the effect of lipoprotein lipase deficiency on the size distribution of fat cell populations in human adipose tissues, abdominal and femoral subcutaneous fat tissue biopsies were obtained from seven patients affected by familial hyperchylomicronaemia. These patients were characterized by massive accumulation of chylomicrons in the fasting state due to defective catabolism of plasma triglyceride-rich lipoproteins. They had no post-heparin plasma lipoprotein lipase activity and their fat tissues were deficient in lipoprotein lipase activity. The size distribution of adipocytes examined by scanning electron microscopy were similar to distributions observed in control subjects. Patient fat cell diameters were not statistically different from control fat cells obtained from subjects of similar body mass index. Mature fat cells contributed to 99% of the total fat tissue mass in lipoprotein lipase deficiency. Normal adiposity in lipoprotein lipase deficiency can thus be attributed to mature adipocytes and not to hyperplastic growth of immature fat cells. It is concluded that normal adipose tissue homeostasis is maintained in these patients in spite of the deficiency in lipoprotein lipase activity.