Open surgery induces a dramatic decrease in circulating intact IGFBP-3 in patients with colorectal cancer not seen with laparoscopic surgery.

BACKGROUND: As shown earlier by the authors via Western blot analysis, open (OS) but not laparoscopic surgery (LS) induces a qualitative decrease in plasma insulin-like growth factor-binding protein 3 (IGFBP-3) levels on postoperative day 1 (POD 1). Intact IGFBP-3 has tumor suppressive effects, but its degradation products do not. Enzyme linked immunoassay (ELISA) inevitably measures both. In this study, using a novel combined Western blot and ELISA analysis method, precise plasma levels of intact IGFBP-3 on POD2 after open and closed colorectal cancer resection (stage I-III) were determined. METHODS: This study included 15 OS patients with a mean incision length of 26.7 +/- 15.5 cm and 16 LS patients with a mean incision length of 5.3 +/- 3.1 cm. Intact IGFBP-3 levels were determined via ELISA and Western blot analysis in plasma collected preoperatively and postoperatively. RESULTS: In the OS patients, the mean preoperative concentration of intact 43-45 kDa IGFBP-3 protein was 1920 +/- 1430 ng/ml. It decreased dramatically on POD2 to 355 +/- 545 ng/ml (p < 0.005). In the LS group, no significant difference was noted between the preoperative level (1305 +/- 807 ng/ml) and the POD2 level (922 + 714 ng/ml). CONCLUSIONS: Open cancer resection, unlike its minimally invasive alternative, induces a dramatic decrease in concentration of intact IGFBP-3, which may have important implications with regard to colon cancer recurrence.

Addition of interleukin-12 to GA733 tumor protein vaccine leads to development of tumor protective immunity despite surgical stress.

BACKGROUND: We have previously shown that preoperative vaccination with the GA733 protein does not inhibit tumor growth in mice undergoing open surgery or carbon dioxide insufflation. In this study we assessed the antitumor effect of a combined GA733 and interleukin-12 (IL-12) vaccine. METHODS: For this study, BALB/c mice were immunized with GA733, IL-12, or GA733 and IL-12, or they received no vaccine. Immediately after surgery (laparotomy or insufflation), GA733-transfected CT26 cells (C26-GA733) were injected subcutaneously into all mice. After 5 weeks, the mice were sacrificed, their tumors measured, GA733-specific antibodies determined by enzyme-linked immunoassay, and GA-733-specific cytotoxicity tested by flow cytometry using labeled C26-GA733 cells. RESULTS: Tumors were significantly (p < 0.05) smaller in both the insufflation and open groups that received combined GA733 and IL-12 than in their respective control subjects. Vaccination also induced a significant increase in the antibody and cell-mediated tumor-specific immunity. CONCLUSION: A preoperative vaccine consisting of GA733 and IL-12 inhibited postoperative tumor growth after open and closed surgery and allowed the mice to overcome the immunosuppressive effects of surgery.

Human parainfluenza virus type 3 HN-receptor interaction: effect of 4-guanidino-Neu5Ac2en on a neuraminidase-deficient variant.

The envelope of human parainfluenza virus type 3 (HPF3) contains two viral glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). HN, which is responsible for receptor attachment and for promoting F-mediated fusion, also possesses neuraminidase (receptor-destroying) activity. We reported previously that 4-guanidino-neu5Ac2en (4-GU-DANA) and related sialic acid-based inhibitors of HPF3 neuraminidase activity also inhibit HN-mediated receptor binding and fusion processes not involving neuraminidase activity. We have now examined this mechanism, as well as neuraminidase's role in the viral life cycle, using a neuraminidase-deficient HPF3 variant (C28a) and stable cell lines expressing C28a or wild-type (wt) HN. C28a, which has a wt F sequence and two point mutations in the HN gene corresponding to two amino acid changes in the HN protein, is the first HPF3 variant with insignificant neuraminidase activity. Cells expressing C28a HN did not bind erythrocytes at 4 degrees C unless pretreated with neuraminidase, but no such pretreatment was required for hemadsorption activity (HAD) at 22 or 37 degrees C. HAD was blocked by 4-GU-DANA, attesting to the ability of this compound to inhibit HN's receptor-binding activity. C28a or wt plaque enlargement, a process that involves cell-cell fusion and does not depend on virion release, is diminished by the presence of 4-GU-DANA, confirming the inhibitory effect of 4-GU-DANA on the fusogenic function of C28a HN. In C28a-infected cell monolayers, virion release and thus multicycle replication are severely restricted. This defect was corrected by supplementation of exogenous neuraminidase and also by the addition of 4-GU-DANA; neuraminidase destroys the receptors whereby newly formed C28a virions would remain attached to the cell surface, whereas 4-GU-DANA prevents the attachment itself, obviating the need for receptor cleavage. In accord with the ability of 4-GU-DANA to prevent attachment, the neuraminidase inhibitory effect of 4-GU-DANA on wt HPF3 did not diminish virion release into the medium. Thus, it is by inhibition of viral entry and syncytium formation that sialic acid analogs like 4-GU-DANA may counteract wt HPF3 infection.

A single amino acid alteration in the human parainfluenza virus type 3 hemagglutinin-neuraminidase glycoprotein confers resistance to the inhibitory effects of zanamivir on receptor binding and neuraminidase activity.

Entry and fusion of human parainfluenza virus type 3 (HPF3) requires interaction of the viral hemagglutinin-neuraminidase (HN) glycoprotein with its sialic acid receptor. 4-Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (4-GU-DANA; zanamivir), a sialic acid transition-state analog designed to fit the influenza virus neuraminidase catalytic site, possesses antiviral activity at nanomolar concentrations in vitro. We have shown previously that 4-GU-DANA also inhibits both HN-mediated binding of HPF3 to host cell receptors and HN's neuraminidase activity. In the present study, a 4-GU-DANA-resistant HPF3 virus variant (ZM1) was generated by serial passage in the presence of 4-GU-DANA. ZM1 exhibited a markedly fusogenic plaque morphology and harbored two HN gene mutations resulting in two amino acid alterations, T193I and I567V. Another HPF3 variant studied in parallel, C-0, shared an alteration at T193 and exhibited similar plaque morphology but was not resistant to 4-GU-DANA. Neuraminidase assays revealed a 15-fold reduction in 4-GU-DANA sensitivity for ZM1 relative to the wild type (WT) and C-0. The ability of ZM1 to bind sialic acid receptors was inhibited 10-fold less than for both WT and C-0 in the presence of 1 mM 4-GU-DANA. ZM1 also retained infectivity at 15-fold-higher concentrations of 4-GU-DANA than WT and C-0. A single amino acid alteration at HN residue 567 confers these 4-GU-DANA-resistant properties. An understanding of ZM1 and other escape variants provides insight into the effects of this small molecule on HN function as well as the role of the HN glycoprotein in HPF3 pathogenesis.

The anti-influenza virus agent 4-GU-DANA (zanamivir) inhibits cell fusion mediated by human parainfluenza virus and influenza virus HA.

4-GU-DANA (zanamivir) (as well as DANA and 4-AM-DANA) was found to inhibit the neuraminidase activity of human parainfluenza virus type 3 (HPF3). The viral neuraminidase activity is attributable to hemagglutinin-neuraminidase (HN), an envelope protein essential for viral attachment and for fusion mediated by the other envelope protein, F. While there is no evidence that HN's neuraminidase activity is essential for receptor binding and syncytium formation, we found that 4-GU-DANA prevented hemadsorption and fusion of persistently infected cells with uninfected cells. In plaque assays, 4-GU-DANA reduced the number (but not the area) of plaques if present only during the adsorption period and reduced plaque area (but not number) if added only after the 90-min adsorption period. 4-GU-DANA also reduced the area of plaques formed by a neuraminidase-deficient variant, confirming that its interference with cell-cell fusion is unrelated to inhibition of neuraminidase activity. The order-of-magnitude lower 50% inhibitory concentrations of 4-GU-DANA (and also DANA and 4-AM-DANA) for plaque area reduction and for inhibition in the fusion assay than for reducing plaque number or blocking hemadsorption indicate the particular efficacy of these sialic acid analogs in interfering with cell-cell fusion. In cell lines expressing influenza virus hemagglutinin (HA) as the only viral protein, we found that 4-GU-DANA had no effect on hemadsorption but did inhibit HA2b-red blood cell fusion, as judged by both lipid mixing and content mixing. Thus, 4-GU-DANA can interfere with both influenza virus- and HPF3-mediated fusion. The results indicate that (i) in HPF3, 4-GU-DANA and its analogs have an affinity not only for the neuraminidase active site of HN but also for sites important for receptor binding and cell fusion and (ii) sialic acid-based inhibitors of influenza virus neuraminidase can also exert a direct, negative effect on the fusogenic function of the other envelope protein, HA.

Mechanism of interference mediated by human parainfluenza virus type 3 infection.

Viral interference is characterized by the resistance of infected cells to infection by a challenge virus. Mechanisms of viral interference have not been characterized for human parainfluenza virus type 3 (HPF3), and the possible role of the neuraminidase (receptor-destroying) enzyme of the hemagglutinin-neuraminidase (HN) glycoprotein has not been assessed. To determine whether continual HN expression results in depletion of the viral receptors and thus prevents entry and cell fusion, we tested whether cells expressing wild-type HPF3 HN are resistant to viral infection. Stable expression of wild-type HN-green fluorescent protein (GFP) on cell membranes in different amounts allowed us to establish a correlation between the level of HN expression, the level of neuraminidase activity, and the level of protection from HPF3 infection. Cells with the highest levels of HN expression and neuraminidase activity on the cell surface were most resistant to infection by HPF3. To determine whether this resistance is attributable to the viral neuraminidase, we used a cloned variant HPF3 HN that has two amino acid alterations in HN leading to the loss of detectable neuraminidase activity. Cells expressing the neuraminidase-deficient variant HN-GFP were not protected from infection, despite expressing HN on their surface at levels even higher than the wild-type cell clones. Our results demonstrate that the HPF3 HN-mediated interference effect can be attributed to the presence of an active neuraminidase enzyme activity and provide the first definitive evidence that the mechanism for attachment interference by a paramyxovirus is attributable to the viral neuraminidase.

IS1245 genotypic analysis of Mycobacterium avium isolates from patients in Brazil.

OBJECTIVE: Disseminated Mycobacterium avium infection is an emerging opportunistic disease among patients with acquired immunodeficiency syndrome (AIDS) in Brazil. The mode of transmission of M. avium in a developing country setting needs to be better characterized. METHODS: Mycobacterium avium strain collections in São Paulo and Rio de Janeiro were analyzed according to the strains' IS1245 DNA gel electrophoretic migration patterns. Medical records of the patients from whom M. avium isolates were available were reviewed, and their demographic characteristics were stratified according to the isolates' IS1245 DNA fingerprint patterns. RESULTS: Of 105 patients, 33 (31%) with M. avium isolated between 1990 and 1994 had strains having IS1245 patterns identical in patterns seen in isolates from two or more patients (designated as cluster pattern strains). Cluster pattern strains were isolated from 21 (39%) of 54 patients with disseminated infection (defined as infection due to M. avium isolated from a sterile site in an adult patient). Six of the cluster pattern strains were isolated only from sterile sites. In São Paulo, cluster pattern strains were significantly more likely to be isolated from patients with disseminated disease. CONCLUSIONS: These preliminary observations suggest that in large cities of Brazil, a high proportion (at least 39%) of disseminated M. avium infections in patients with AIDS results from a recent transmission. Some strains of M. avium may be more likely to cause disseminated disease than others after an infection.

Molecular epidemiology of tuberculosis in Prague: analysis by restriction fragment length polymorphism.

OBJECTIVES: To characterize by restriction fragment length polymorphism (RFLP) patterns, the distribution of different Mycobacterium tuberculosis strains isolated consecutively from 75 tuberculosis patients who resided in Prague and had culture-confirmed cases during a 4-month period in 1995. METHODS: The insertion sequence IS6110-based RFLP analysis of M. tuberculosis isolates was carried out. RESULTS: There were a total of 75 patients with various forms of tuberculosis (54 males; 21 females). The sources of M. tuberculosis isolates were sputum (n = 64), pleura or lymph node drainage (n = 8), and urine (n = 3). Fifty-three of the patients (70.7%) had isolates with unique RFLP patterns, while 22 (29.3%) had isolates that belonged to seven clusters of related RFLP patterns. The seven clusters consisted of four groups of two patients, two groups of four patients, and one group of six patients. Most of the patients whose isolates fell within a clustered RFLP pattern lived in different quarters of the city and had no identifiable contacts with other patients whose isolates had the same pattern. CONCLUSIONS: The finding that isolates from most patients (70.7%) had unique rather than clustered RFLP patterns suggests that endogenous reactivation rather than exogenous transmission is the major determinant of most of the tuberculosis cases in Prague. The occurrence of seven distinct clusters comprising 29.3% of the isolates suggests that approximately one third of cases developed active tuberculosis from recent exogenous transmission.

Widespread dissemination of a drug-susceptible strain of Mycobacterium tuberculosis.

In New York City, a large proportion of new tuberculosis cases has been caused by 1 drug-susceptible strain (called C strain) of Mycobacterium tuberculosis. Between 1991 and 1994, among >600 tuberculosis patients consecutively identified in four large hospitals in the city, 54 with C strain, 69 with non-C cluster pattern strains, and 42 with noncluster pattern strains were studied. Susceptibility to reactive nitrogen intermediates (RNI) of selected isolates was compared. In a case-control analysis, 51% of patients with C strain, 28% with non-C cluster strains (P < .05), and 14% with noncluster strains (P < .01) were found to be injection drug users. C strain but not 13 other unrelated isolates were resistant to RNI. Injection drug use may provide a selective pressure for an RNI-resistant tubercle bacillus to emerge, which may give the organism a biologic advantage and explain the widespread dissemination of C strain M. tuberculosis within the city.

Tuberculosis among foreign-born persons in New York City, 1992-1994: implications for tuberculosis control.

OBJECTIVE: To study the pattern of transmission of tuberculosis (TB) among foreign-born persons living in New York City. DESIGN: A retrospective multicenter study comparing 158 foreign-born patients to 231 US-born patients diagnosed with TB between 1992 and 1994. The patients were stratified according to their Mycobacterium tuberculosis isolate DNA fingerprint patterns. RESULTS: Nineteen (16%) of 122 isolates from foreign-born TB patients and 75 (42%) of 180 isolates from US-born TB patients had DNA fingerprint patterns (cluster patterns) indicative of recent exogenous transmission (P < 0.001). All cluster pattern strains from foreign-born cases were identical to those found among US-born patients. The likelihood of infection with a cluster pattern strain among foreign-born persons increased with duration of residence in the US, and was significantly associated with being homeless (P < 0.05), or having multidrug-resistant TB (P = 0.00072). CONCLUSION: Although most (84%) cases of TB among foreign-born persons in New York City appear to result from reactivation of infections they acquired abroad, the ones who acquire new infections become infected with strains that are already circulating among the US-born TB patients in New York City, and they have risk factors similar to those faced by US-born tuberculosis patients.

High sensitivity of Mycobacterium species to the bactericidal activity by polylysine.

Bactericidal effects of polylysine on different bacterial species were measured. Marked differences in sensitivity were observed. Based on the concentration of polylysine required to reduce cell viability by 50%, Mycobacterium smegmatis and Mycobacterium tuberculosis were found to be the most sensitive and Escherichia coli the most resistant. In addition, two Gram-positive organisms, Staphylococcus epidermidis and Streptococcus salivarius exhibited significant differences in sensitivity which suggests that the relationship between sensitivity towards polylysine and bacterial cell type is not necessarily a function of the overall cell envelope structure. The high sensitivity of mycobacteria suggests the possible use of polylysine, or a conjugate of polylysine and another agent in anti-mycobacterial drug design.