Secretory leukocyte protease inhibitor: a human saliva protein exhibiting anti-human immunodeficiency virus 1 activity in vitro.

Infection of adherent primary monocytes with HIV-1Ba-L is significantly suppressed in the presence of human saliva. By reverse transcriptase (RT) levels, saliva, although present for only 1 h during monocyte viral exposure, inhibited HIV-1 infectivity for 3 wk after infection, whereas human plasma and synovial fluid failed to inhibit HIV-1 infectivity. Antiviral activity was identified in the saliva soluble fraction, and to determine the factor(s) responsible, individual saliva proteins were examined. Of those proteins examined, only secretory leukocyte protease inhibitor (SLPI) was found to possess anti-HIV-1 activity at physiological concentrations. SLPI anti-HIV-1 activity was dose dependent, with maximal inhibition at 1-10 micrograms/ml (> 90% inhibition of RT activity). SLPI also partially inhibited HIV-1IIIB infection in proliferating human T cells. SLPI appears to target a host cell-associated molecule, since no interaction with viral proteins could be demonstrated. However, SLPI anti-HIV-1 activity was not due to direct interaction with or downregulation of the CD4 antigen. Partial depletion of SLPI in whole saliva resulted in decreased anti-HIV-1 activity of saliva. These data indicate that SLPI has antiretroviral activity and may contribute to the important antiviral activity of saliva associated with the infrequent oral transmission of HIV-1.

Secretory leukocyte protease inhibitor suppresses the production of monocyte prostaglandin H synthase-2, prostaglandin E2, and matrix metalloproteinases.

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor found in fluids lining mucosal surfaces. In addition to its primary function as an antiprotease, SLPI may also influence cellular functions associated with enzyme synthesis and retroviral infection. In this study, SLPI was examined for its effect on signaling events involved in the production of matrix metalloproteinases (MMPs) by monocytes. Addition of SLPI before stimulation with concanavalin A or LPS resulted in a significant inhibition of monocyte prostaglandin H synthase-2 (PGHS-2), a pivotal enzyme in the PGE2-cAMP dependent pathway of monocyte MMP synthesis. Suppression of PGHS-2 was detected with 0.1 microg/ml of SLPI with a substantial inhibition at 1 and 10 micro/ml. Attenuation of PGHS-2 by SLPI was accompanied by decreased production of PGE2 resulting in the suppression of interstitial collagenase (MMP-1) and gelatinase B (MMP-9) that was reversed by PGE2 or Bt2cAMP. The inhibitory effect of SLPI was largely independent of its antiprotease activity because SLPI muteins, with significantly lower antiprotease activity, also suppressed the induction of PGHS-2 and MMPs. The inhibitory effects of SLPI did not involve the modulation of monokine production since TNF-alpha and IL-10 were unaffected. These findings demonstrate that SLPI also functions as a potent antiinflammatory agent by interfering with the signal transduction pathway leading to monocyte MMP production.

Stable DNA transfection of a wide range of trypanosomatids.

We have shown that the Leishmania major transfection vector pR-NEO (or derivatives thereof) can be introduced and stably maintained in four species complexes of pathogenic Leishmania (L. tropica, L. mexicana, L. donovani, L. braziliensis), and the genera Endotrypanum and Crithidia; transfection of Trypanosoma cruzi or Trypanosoma brucei was not successful. Quantitative plating assays showed that the transfection efficiencies were high in L. major and Leishmania amazonensis (5x10(-5)/cell) and about 10-fold less for Leishmania panamaensis and Crithidia. Leishmania donovani transfected with pR-NEO retained the ability to infect hamsters, and amastigotes recovered after 2 months yielded G418-resistant promastigotes which retained high levels of extrachromosomal pR-NEO DNA. In promastigotes, the transfected DNA existed as extrachromosomal circles, and expressed the predicted 2.4-kb hybrid NEO/DHFR-TS mRNA bearing the trans-spliced miniexon. Large quantitative differences were observed only in Crithidia: relative to transfected Leishmania species, the copy number of pR-NEO was elevated 20-fold, while the levels of the NEO/DHRFR-TS mRNA or Escherichia coli beta-galactosidase (synthesized from the expression vector pX-beta GAL) were reduced 80 and more than 1000-fold, respectively. Thus, genetic signals derived from L. major DNA that mediate RNA expression or stability are recognized by the heterologous Leishmania species but less efficiently by Crithidia. These studies suggest that pR-NEO derived vectors may be applied to the study of genes expressed throughout the life cycle in a wide range of pathogenic trypanosomatids.

Defect of insulin receptor in insulin-resistant variants of Cloudman S91 mouse melanoma cells.

To study the mechanisms by which insulin regulates proliferation, we have compared wild-type Cloudman melanoma cells, whose growth in inhibited by insulin (insinh) to variant lines that were genetically selected for resistance to insulin (insres). Scatchard analysis of insulin binding to five insres) lines and six insres variants revealed a marked reduction in the number of high-affinity binding sites for insulin in the insres lines, and insres lines displayed an abnormal beta-subunit of the insulin receptor. During autophosphorylation of the wheat germ agglutinin-purified receptor, the beta-subunit apparently underwent proteolytic degradation. This proteolysis was ATP-dependent and was prevented by bovine pancreatic trypsin inhibitor, and phenylmethylsulphonyl fluoride, but not by aprotinin or leupeptin. Receptor proteolysis was not observed in wild-type lines. The results suggest that insulin resistance in the mutant Cloudman melanoma cells is apparently due to proteolysis of the beta-subunit of insulin receptor which, in turn, alters insulin binding capacity of the cells and blocks their anti-proliferative response to the hormone.

Acantholytic variant of squamous cell carcinoma of the breast: a case report.

Primary squamous cell carcinoma of the breast is a rare clinical entity. Two large review series found only five cases out of a total of 8351 breast malignancies. This case report presents a patient with metaplastic, pseudoangiosarcomatous carcinoma or acantholytic variant of a squamous cell carcinoma of the breast. This diagnosis was based on the histological finding of highly atypical, acantholytic squamous cells. Because the tumor stained positive for keratin and negative for factor VIII, the diagnosis of angiosarcoma was ruled out. Although only scattered case reports have been published on this histological variant, these tumors tend to follow an aggressive course.

Angiokeratoma of the clitoris.

A 25-year-old woman, multigravida, presented with a dark ulcerated tumor of the clitoris. Histologic examination demonstrated an angiokeratoma, a benign telangiectatic vascular tumor. To our knowledge, this is the first report of angiokeratoma of the clitoris and only the fifth describing vascular tumor of this organ. The clinical differential diagnosis of angiokeratoma and of clitoral tumors includes malignant neoplasms such as melanoma. Biopsy with histologic examination is, therefore, recommended to ensure appropriate treatment of these unusual tumors.

Pericellular lacunae in effusions.

Pericellular lacunae are reported to be characteristic of adenocarcinoma cell clusters in cell block preparations of serous effusions. We examined the incidence of lacunae in cell blocks from 24 benign and 31 malignant effusions. The cases were classified as follows: group A with no pericellular lacunae, group B with < 50% of cell clusters in lacunae, and group C with > 50% of cell clusters in lacunae. Of the malignant effusions, 5% were in group A, 67% were in group B and 29% were in group C. Of the benign effusions, 50% were in group A, 50% were in group B, and 0% were in group C. The pericellular lacuna by itself is not a reliable diagnostic criterion. It could assist, however, in the overall evidence when evaluating a case, particularly if either there are no lacunae seen or the majority of cells are in lacunae. Furthermore, since lacunae are easily visible at low magnification, their recognition may help in screening cell blocks, using their presence to locate cell groups worthy of special attention.

Diagnosis of follicular lymphoma by fine needle aspiration biopsy.

This study evaluated the diagnostic accuracy of fine needle aspiration biopsy (FNAB) of follicular lymphoma (FL). Fourteen aspirates of lymph nodes in which follow-up surgical biopsy revealed FL were studied. Two aspirates were deemed unsatisfactory because of a paucity of cells. The remaining 12 cases received the following diagnoses: 4 positive for malignant lymphoma, 4 highly suspicious for malignant lymphoma and 4 false negatives. FNAB of FL can show a monomorphic or polymorphic cell population. The aspirates with a positive or suspicious diagnosis showed monomorphic cell populations. False-negative diagnoses were attributable to misleading sampling or preparation methods in most cases. We conclude that FNAB of FL is less accurate than FNAB of non-Hodgkin's malignant lymphoma (NHL) in general, but the accuracy rate is similar to that of FNAB of all low-grade NHL. The value of current approaches to the diagnosis of suspected lymphoma by FNAB is emphasized.

Comparison of the opsonic activity of human surfactant protein A for Staphylococcus aureus and Streptococcus pneumoniae with rabbit and human macrophages.

Surfactant protein A (SP-A) is a glycosylated apoprotein that may facilitate bacterial phagocytosis and contribute to early bacterial clearance in the lung. The effect of SP-A on attachment (or ingestion) of Staphylococcus aureus and type 25 pneumococci to rabbit alveolar macrophages and human monocyte-derived macrophages was studied. SP-A bound to S. aureus and type 25 pneumococci in a calcium-dependent manner. Bacteria-associated SP-A significantly increased attachment of S. aureus, but not pneumococci, to macrophages. Increased association of SP-A-coated S. aureus with macrophages appeared to consist mainly of attachment without ingestion, as determined by bactericidal tests and release of tritiated bacterial digestion products from macrophages. Preincubation of macrophages with SP-A did not increase attachment or ingestion of S. aureus or type 25 pneumococci, with or without the addition of immune opsonins. SP-A acts as a ligand to facilitate attachment of S. aureus to macrophages but has no effect on S. pneumoniae.

Aggregation and opsonization of type A but not type B Hemophilus influenzae by surfactant protein A.

The ability of surfactant protein A (SP-A) to aggregate and opsonize type a and b Hemophilus influenzae was investigated. Type a, but not type b, was aggregated by SP-A. Aggregation was maximal at 24 micrograms SP-A/ml and was Ca(2+)-dependent. Aggregation of type a was inhibited by D-glucosyl-BSA but not by high concentrations of monosaccharides (D-mannose, D-galactose, D-glucose, or L-fucose) or by sialic acid, purified type a capsular polysaccharide, or type IV collagen. In Western blots, 125I-labeled SP-A bound to the major outer membrane protein (putatively P2) of type a hemophilus by a Ca(2+)-dependent mechanism. This binding was competitively inhibited by excess unlabeled SP-A. 125I-labeled SP-A also bound to the major membrane protein of type b, but at less than 5% of the level observed for type a. SP-A did not bind to lipooligosaccharides of either type a or type b. SP-A increased association of type a, but not type b, hemophilus with alveolar macrophages. After opsonization with SP-A, type a hemophilus were killed by alveolar macrophages, as indicated by bactericidal assays and the release of soluble, radiolabeled products from leukocytes. It is concluded that SP-A aggregated and opsonized type a hemophilus, but not type b, possibly because SP-A bound to the P2 outer membrane protein of type a to a greater extent.

Isolation of lipophosphoglycans from Leishmania donovani amastigotes.

Leishmania donovani donovani amastigotes, isolated from spleens of infected hamsters or axenically cultured, and promastigotes were comparatively examined for the expression of lipophosphoglycans (LPG). Parasites were metabolically labeled with [32p]-phosphate, [3H]galactose, or [3H]mannose. Radiolabeled material was extracted with water/ethanol/diethylether/pyridine/NH40H and purified further by gel filtration and hydrophobic column chromatographies. These radiolabeled compounds were identified as phosphorylated lipid-containing glycoconjugates. Mild acid treatment resulted in degradation of the glycolipids into low-molecular-weight fragments. All glycolipids isolated were susceptible to nitrous acid and phosphatidylinositol-specific phospholipase C treatments, as has been reported for the L. donovani promastigote LPG. Moreover, glycoconjugates purified from the three Leishmania stocks were not susceptible to trypsin treatment. Acid hydrolysis of promastigote LPG resulted in a predominant [P04-6galactose(beta1,4)mannose(alpha)1] fragment. In contrast, the main radiolabeled anionic fragments isolated from splenic and axenic amastigotes differed from that of promastigotes, as evidenced by the elution profiles obtained by HPLC anion-exchange chromatography. These cumulative results indicate that lipophosphoglycan molecules, structurally distinct from the previously characterized LPG of the promastigote stage, are being expressed by L. donovani splenic and axenic amastigotes.

Adrenal cortical cells mimicking small cell anaplastic carcinoma in a fine-needle aspirate.

Fine-needle aspiration of an adrenal mass was performed to rule out metastatic disease in a patient with a bronchogenic carcinoma. The aspiration was misinterpreted as metastatic small cell anaplastic carcinoma. Review of the aspirate by the authors revealed the atypical small cells to be aggregates of bare nuclei of benign adrenocortical cells. Interpretation of these bare nuclei in isolation and unfamiliarity with the aspiration cytology of the adrenal gland led to the initial misdiagnosis.

The anticoagulant mechanism of action of heparin in contact-activated plasma: inhibition of factor X activation.

The effects of heparin on the activation of blood coagulation factors IX and X in contact-activated plasma were determined in the present study. In the presence and absence of 0.5 U/mL heparin, the amounts of factor IX that were cleaved 30 minutes after the addition of calcium and phospholipid to plasma exposed to glass (ie, contact activated) were essentially identical. In the absence of heparin, however, the plasma clotting time was between three and four minutes, while in the presence of heparin, the clotting time was approximately 40 minutes. More factor IXa was inhibited by antithrombin III in the presence of heparin than in its absence, but factor IXa levels sufficient for factor X activation appeared to be present in the heparinized plasma. Neither an increase in factor Xa nor a decrease in factor X was detected, however, in heparinized plasma. We conclude that the step in the intrinsic pathway of coagulation that is inhibited in the presence of heparin is at the level of factor X activation.

Inhibition of protein kinase C activity by the Leishmania donovani lipophosphoglycan.

Purified lipophosphoglycan from Leishmania donovani was found to inhibit the activity of protein kinase C isolated from rat brain. Protein kinase C inhibition by lipophosphoglycan was continuous for 30 minutes. The glycoconjugate was a competitive inhibitor with respect to diolein, a noncompetitive inhibitor with respect to phosphatidylserine, and had no significant effect on protein kinase M and protein kinase A. A possible physiological role of lipophosphoglycan as a negative effector of protein kinase C is suggested.

Requirement of lipophosphoglycan for intracellular survival of Leishmania donovani within human monocytes.

The function of the lipophosphoglycan of Leishmania donovani parasites was investigated in human peripheral monocytes. In contrast to wild-type L. donovani which grow in monocytes, incubation of monocytes with two variant lines of L. donovani, defective in lipophosphoglycan expression, resulted in the entry of the variant cells into the monocytes and their subsequent destruction. Passive transfer of lipophosphoglycan to the variant cells led to prolonged survival in monocytes. These results indicate that lipophosphoglycan is required by the parasite for intracellular survival. To investigate one possible protective role of the glycoconjugate, preincubation of monocytes with a suspension of lipophosphoglycan and subsequent treatment of the cells with PMA or opsonized zymosan resulted in an attenuation of the oxidative burst; the attenuation effect was concentration dependent on the glycoconjugate and independent of preincubation time. Moreover, hydrophobic beads, coated with lipophosphoglycan, were phagocytized by monocytes and found to inhibit oxygen consumption in monocytes activated with PMA. These results suggest a possible relationship between the absence of lipophosphoglycan in the variant parasites and their inability to survive within monocytes. Although the precise molecular basis remains to be elucidated, the ability of lipophosphoglycan to impair the microbial oxidative response may be a contributing factor in its requirement for intracellular survival.

Effect of individual conjugate dose on immunogenicity of type 6B pneumococcal polysaccharide-N. meningitidis outer membrane protein complex conjugate vaccines in infant rhesus monkeys.

A pneumococcal conjugate vaccine (PCV) has been developed consisting of capsular polysaccharide (Ps) coupled to the outer membrane protein complex of Neiserria meningitidis serogroup B. Experiments were conducted in infant rhesus monkeys to assess the potential to administer multiple Pn types in a single vaccine. A single type conjugate, 6B, was dosed from 0.025 to 25 microg Ps. Peak anti-6B Ps Ab titers were seen at lower doses of 0.025 and 0.25 microg Ps, while reduced titers of anti-6B Ps Ab were observed at the highest doses of conjugate administered, 2.5 and 25 microg Ps. By mixing free Ps, carrier, or another monovalent PCV with this 6B PCV, it was determined that reduced anti-6B Ps titers at high PCV doses were associated only with the quantity of type-specific Ps in the conjugate. Thus, increasing the amount of carrier protein or adding an additional monovalent conjugate did not significantly affect the response to type 6B Ps. These results suggest that, given an appropriately determined dose per individual pneumococcal Ps type, a multivalent PCV that includes many different types should have satisfactory clinical immunogenicity.

Inhibitory effects on protein kinase C activity by lipophosphoglycan fragments and glycosylphosphatidylinositol antigens of the protozoan parasite Leishmania.

Fragments of the lipophosphoglycan of Leishmania donovani were generated by phospholipase C digestion and mild acid hydrolysis. The fragments were purified and examined for inhibitory activity on protein kinase C isolated from rat brains. On a molar basis, the 1-O-alkylglycerol portion of LPG exhibited the most inhibitory activity, whereas the carbohydrate domain was not as effective. In addition, several glycolipid antigens from L. major, which contain short carbohydrate chains attached to phosphatidylinositol, were also efficient inhibitors of the enzyme. These results are consistent with the hypothesis that protein kinase C may be a key target for the parasites to overcome within host macrophages.

Characterization of Leishmania donovani variant clones using anti-lipophosphoglycan monoclonal antibodies.

Monoclonal antibodies directed against the lipophosphoglycan of Leishmania donovani were used to characterize two glycosylation variants of the parasite. One of the variants was found to be totally deficient in the synthesis and expression of lipophosphoglycan. The other variant synthesized lower levels (20%) of the glycoconjugate compared to wildtype cells and its lipophosphoglycan was smaller in size. The two lipophosphoglycan-deficient clones will be useful for elucidating the biosynthesis and function of the glycoconjugate.