The molecular basis of the hyaluronic acid-mediated stimulation of granulocyte function

Previous investigations have demonstrated that the function of polymorphonuclear leukocytes (PMN) is stimulated by hyaluronic acid (HA). The aim of the present investigation was to study the molecular basis for the effect of HA. HA fragments of m.w. in the range from 792 (tetrasaccharide) to 3,000,000 all stimulated the chemotactic and phagocytic function of PMN. The active concentration ranged from 4 to 64 pmol/liter, irrespective of the molecular size. Further investigations demonstrated that N-acetyl-D-glucosamine (NAGA) was the smallest active fragment of HA. NAGA is one of the components from which HA is built up; the other component glucuronic acid was without effect, and so were the other glycosaminoglycans, N-acetyl-D-mannosamine, N-acetyl-D-galactosamine, and D-glucosamine. Finally, Con A, the glucopyranosyl and glucomannosyl binding lectin, inhibited the stimulatory effect of NAGA. As is the case with HA, fibronectin also acts as a necessary cofactor to NAGA when incubations are made in the absence of whole blood or serum. The present results strongly indicated that the combined action of NAGA and fibronectin worked directly on the PMN by an interaction at the cellular membrane level. We conclude that the stimulatory action of HA on granulocyte functions is mediated through one of its two structural components, i.e., NAGA.     

Acquisition of non-MHC restricted cytotoxic function by IL 2 activated thymocytes with an "immature" antigenic phenotype

Culture of human thymocytes in interleukin 2 (IL 2) results in the generation of cytotoxic T lymphocytes (CTL) that kill tumor cell targets without major histocompatibility complex (MHC) restriction. Thymic non-MHC restricted CTL expressed Leu-19 antigen, but were generated from thymic precursor cells that lacked expression of Leu-19. In contrast, short term culture in Il 2 of peripheral blood lymphocytes depleted of Leu-19+ lymphocytes did not result in the generation of cytotoxic activity. IL 2 was necessary and sufficient for the generation of cytotoxic thymocytes and induction of Leu-19 antigen expression. Thymic non-MHC restricted CTL were generated from precursor cells expressing CD1, an antigen present on the majority of thymocytes. Furthermore, cytotoxic activity was detected in IL 2 cultured thymocyte populations with an "immature" antigenic phenotype, i.e. CD1+ and CD4+, CD8+. Upon subsequent culture, thymic non-MHC restricted CTL lost expression of CD1, and developed an antigenic phenotype similar to peripheral blood non-MHC-restricted CTL, suggesting that peripheral non-MHC-restricted CTL may originate from these thymic precursors.     

Evidence for negative regulation of T cell growth by low affinity interleukin 2 receptors

Two experimental situations have been studied, and the results provide evidence for a negative regulatory role for the low affinity interleukin 2 receptor (LA-IL 2R). The IL 2-dependent T helper cell line L-14, deprived of IL 2, becomes quiescent and expresses comparable numbers of high affinity IL 2R (HA-IL 2R) and LA-IL 2R. After activation by recombinant IL 2, this cell line preferentially expresses LA-IL 2R. The IL 2 responsiveness of the L-14 cell line was found to vary according to the ratio of LA-IL 2R to HA-IL 2R: the relative predominance of the LA-IL 2R coincides with a hyporeactivity of cells to IL 2. In contrast, a predominance of HA-IL 2R is accompanied by an increase in cellular IL 2 reactivity. Treatment of three IL 2-dependent T cell lines (L-14, HT-2, and C30.1) with limited amounts of recombinant IL 2 and moderate concentrations of anti-IL 2R monoclonal antibodies stimulates T cell growth. This treatment was shown to selectively diminish the expression of membrane LA-IL 2R. The stimulation was attributed to the decrease of expression of LA-IL 2R.     

A synthetic peptide homologous to the envelope proteins of retroviruses inhibits monocyte-mediated killing by inactivating interleukin 1

The synthetic peptide CKS-17 has homology to a highly conserved region of the immunosuppressive retroviral envelope protein P15E, to envelope proteins of HTLV I, II, III, and to that encoded by an endogeneous C-type human retroviral DNA. CKS-17 inhibits the immune response of lymphocytes and the respiratory burst of human monocytes. Because P15E-related antigens are present in human malignant cell lines and cancerous effusions, we sought to determine the effect of CKS-17 on monocyte-mediated tumor cell lysis. Lysis of A375 tumor cells by lymphokine or lipopolysaccharide-activated human monocytes was inhibited by 10 microM CKS-17 (control, 79%; CKS-17-treated, 19%). Another synthesized peptide, CS-2, which has partial homology to CKS-17, failed to block monocyte-mediated killing. Thus, the inhibition by CKS-17 appeared to be specific. Because interleukin 1 (IL-1) is a cytocidal factor produced by activated monocytes, we also investigated the effect of CKS-17 on IL-1 production by monocytes and on direct IL-1-mediated cytotoxicity. CKS-17 did not block production or secretion of IL-1 by lipopolysaccharide- or interferon-gamma-activated monocytes. However, the direct cytocidal activity of both recombinant IL-1 alpha and IL-1 beta against A375 tumor cells was blocked by CKS-17. The cytotoxic activity of IL-1 was inhibited by CKS-17 if (a) IL-1 was preincubated with CKS-17 for 1 hr at 37 degrees C or (b) the A375 cells were incubated with CKS-17 for 1 hr prior to the addition of IL-1. CKS-17 also blocked IL-1-induced proliferation of murine thymocytes, the D10 T cell line, and an IL-1-responsive astrocytoma cell line. These data suggest that CKS-17 may be a potent inhibitor of IL-1.     

Retrospective evaluation of gynecologic cytodiagnosis. II. Interlaboratory reproducibility as shown in rescreening large consecutive samples of reported cases

Two laboratories exchanged and rescreened a large sample of cases with cervicovaginal smears they had consecutively accessioned to examine the reproducibility of gynecologic cytodiagnosis under optimum conditions. At least a "working agreement" (diagnoses within +/- 1 category on a ten-category scale) was achieved in diagnoses of normal, benign reaction and squamous abnormality (from minimal dysplasia though invasive cancer) in 18,859 cases (96.8%), of endometrial abnormality in 21 cases (42%) and of "unsatisfactory" in 99 cases (20.7%). Larger differences occurred in greater than or equal to 30% of cases except in the categories of "normal" and "benign reaction," reaching a maximum of 82% for moderate dysplasia. Reexamining 382 cases decreased disagreement by category to the 20% to 65% range only in the five categories of dysplasia plus carcinoma in situ. Agreement was not predicated on the presence of endocervical cells or squamous metaplasia; the basis for "unsatisfactory" calls was not uniform. Comparison of the laboratories' diagnoses with referee diagnoses or, on 178 cases, with tissue diagnoses also demonstrated differences in diagnostic criteria.     

Inflammatory atypia and the false-negative smear in cervical intraepithelial neoplasia

The effectiveness of cervical cytologic screening is compromised by the increasingly recognized prevalence of false-negative smears. Our previous studies suggested that some false-negative cytologies can be accounted for by smears showing cervical intraepithelial neoplasia (CIN) reported as inflammatory atypia; we found that at least 4% of 5,752 consecutive smears had been underreported in this manner. In the present study, that data was reanalyzed to derive 95% confidence limits for the number of CIN smears reported as inflammatory atypia. Using several differing estimates of cytologic screening sensitivity, it is speculated that, under certain testable assumptions, colposcopy of patients with cytologic diagnoses of inflammatory atypia may be one cost-effective approach to finding CIN cases missed by screening. If confirmed, these findings imply that laboratory quality assurance efforts, traditionally directed to the most serious cytologic diagnoses, should also focus in part on nondysplastic atypia.     

Luteal morphology, atresia, and plasma progesterone concentrations during the reproductive cycle of two oviparous lizards, Crotaphytus collaris and Eumeces obsoletus

From ovulation to oviposition, the corpora lutea of the oviparous lizards Crotaphytus collaris and Eumeces obsoletus exhibit three stages of luteal development: 1) luteogenesis, 2) luteal maturity, and 3) luteal regression. Each stage exhibits distinct characteristics, involving changes in: 1) luteal volume, 2) nuclear diameter of cells within the luteal cell mass, and 3) thecal development. Plasma progesterone concentration is greatest during luteogenesis and is positively correlated with ovarian atresia, although atresia occurred throughout the period of gravidity. These data suggest that in these two species, the corpora lutea secrete high amounts of progesterone immediately following ovulation and exhibit morphologically distinct stages of growth and regression.     

Radioreceptor assay in checking serum concentration in long-term treatment with cis(z)-flupenthixol decanoate

24 patients have been treated with cis(z)-flupenthixol decanoate for 6-12 months. Intramuscular injections were given about every 3 weeks. Before treatment and on each day of injection the mental state was assessed by BPRS and registration of side effects was performed. Blood samples were taken 7 days after each injection and on the last day of the dosage interval. Neuroleptic activity was determined in serum by RRA and expressed in cis(z)-flupenthixol equivalents. The drug level was significantly correlated to the dose. No clear relationship between drug level and clinical results as well as side effects was found. Less pronounced variations of the drug level between subsequent injections resulted in a positive therapeutic response.     

Determination of small quantities of sulfate (0-12 nmol) in serum, urine, and cartilage of the mouse

The colorimetric benzidine method of K. S. Dodgson and B. Spencer (1953, Biochem. J. 55, 436-440) for the measurement of inorganic sulfate can be scaled down about 100 times by using disposable 96-well microplates instead of individual cuvettes. Ten-microliter samples of serum and urine, derived from mice, can be analyzed in a simple, rapid, and reliable way without sacrificing the animals. Without prior isolation of sulfated glycosaminoglycans, ester sulfate in mouse patellar cartilage is liberated quantitatively as inorganic sulfate upon acid hydrolysis in 3 M HCl for 16 h at 80 degrees C. To this end the articular cartilage layer of the patella must be separated in toto from the underlying bone. Subsequent hydrolysis in polypropylene tubes gives accurate results. In contrast, hydrolysis in borosilicate glass vials is useless, since nanomoles of sulfate added cannot be recovered adequately. The thin patellar cartilage layer obtained from 10-week-old male mice contains about 5 nmol of sulfate, an amount easily measured with the developed microplate benzidine method.     

An nptI-sacB-sacR cartridge for constructing directed, unmarked mutations in gram-negative bacteria by marker exchange-eviction mutagenesis

A technique for marker exchange-eviction mutagenesis that enables the construction of directed, unmarked mutations in Gram-negative bacteria was demonstrated in Erwinia chrysanthemi. The technique employs an nptI-sacB-sacR cartridge that is carried on a 3.8-kb BamHI fragment and confers kanamycin (Km) resistance and sucrose sensitivity (due to the production of levansucrase by sacB) in E. chrysanthemi. The cartridge was inserted into a Sau3A site in a cloned E. chrysanthemi pelC gene (encoding pectate lyase isozyme PLc) and then introduced into the Erwinia genome by gene exchange recombination. The resulting mutant was KmR, sucrose-sensitive, and PLc-deficient. The cartridge was then excised from the plasmid-borne pelC gene by PstI cleavage to leave a 28-bp frame-shifting insertion. The pelC allele containing the 28-bp insertion was exchanged for the chromosomal allele containing the nptI-sacB-sacR cartridge by selection for sucrose tolerance. The resulting E. chrysanthemi mutant was Kms and PLc-deficient. The technique permits the construction of complex strains with many directed mutations without the introduction of a corresponding number of antibiotic resistance markers and should prove useful, for example, in exploring the role of the multiple pel genes in E. chrysanthemi.