Changes in vancomycin-resistant Enterococcus faecium causing outbreaks in Brazil.

Enterococci have been implicated in severe human infections as a consequence of associated determinants of virulence and antimicrobial resistance. The majority of vancomycin-resistant Enterococcus faecium (VRE(fm)) connected to outbreaks worldwide pertains to the clonal complex 17 (CC17). In Brazil, the majority of VRE(fm) involved in outbreaks reported so far are not related to CC17. VRE(fm) strains responsible for an outbreak and sporadic cases in hospitals located in the city of Campinas, Brazil, were compared to other VRE(fm) strains in the country. Twenty-two out of 23 E. faecium were vancomycin-resistant and harboured the vanA gene. One vancomycin-susceptible E. faecium (VSE(fm)) strain was included in this study because it was isolated from a patient who one week later harboured a VRE(fm). All strains, except VSE, showed the same alteration in the VanA element characterised by deletion of the left extremity of the transposon and insertion of IS1251 between the vanS and vanH genes. Genes codifying virulence factors such as collagen-adhesin protein, enterococcal surface protein and hyaluronidase were detected in the VRE(fm) and VSE(fm) studied. Both pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) revealed that VRE(fm) and VSE(fm) strains have a clonal relationship. New sequence types (STs) were identified by MLST as ST447, ST448, ST478 and ST412 but all belonged to the CC17. The present study revealed that VRE(fm) outbreaks in Brazil were caused by strains that did not share a common evolutionary history, and that VRE(fm) strains belonging to CC17 could be predominant in Brazil as in other countries.

Determinants of beta-lactam resistance in meningitis-causing Enterobacteriaceae in Brazil.

This study analyzed resistance determinants in extended-spectrum beta-lactamase (ESBL)-producing enterobacteria and the epidemiology of 11 Escherichia coli isolates obtained from meningitis patients in a region of Brazil from 2000 to 2005. ESBL-encoding genes and their genetic environment were investigated by PCR and sequencing. The gene blaCTX-M-2 was identified in 3 different enterobacteria (E. coli, Serratia marcescens, and Proteus mirabilis) downstream of the insertion sequence ISCR1 (localized in class 1 integrons), but not as part of the resistance cassettes region. Multilocus sequence typing (MLST) was used to investigate genetic relationships between the 11 E. coli isolates in this study and strains associated with meningitis in the E. coli MLST database. MLST analysis indicated high genetic diversity among isolates, and no significant genetic relationship was identified with meningitis-causing E. coli in the database. The results in this report reinforce the need to be attentive to meningitis suspected to be due to ESBL-producing enterobacterial isolates, especially where ESBL epidemiology is well known.

First report of vancomycin-resistant staphylococci isolated from healthy carriers in Brazil.

Reduced susceptibility or resistance to vancomycin has been reported among clinical isolates of staphylococci in previous studies. In the present study we report on the isolation of four vancomycin-resistant staphylococcal strains from healthy carriers inside and outside the hospital environment. These carriers did not receive treatment with any antibiotic. All coagulase-negative staphylococcal strains showed variable levels of resistance to several antimicrobial agents, including oxacillin, and unstable resistance to vancomycin, with decreased vancomycin MICs (<4 mg/liter) after 10 days of passage in a nonselective medium. However, exposure of these revertants to vancomycin selected staphylococcal strains resistant to vancomycin at very high frequencies (10(-2) and 10(-3)). The vancomycin resistance in these staphylococcal strains was not mediated by the van gene. The cell wall of the staphylococcal strains studied became thickest after culture in medium containing vancomycin, and the differences in cell wall thickness were statistically significant (P < 0.001). Thus, the thickening of the cell wall in these staphylococcal strains may be an important contributor to vancomycin resistance.

Phase IB trial for malignant melanoma using R24 monoclonal antibody, interleukin-2/alpha-interferon.

The inflammatory tumor lymphocytic infiltrates and spontaneous tumor regressions seen in patients with metastatic malignant melanomas suggest a cellular immune involvement. Enhancement of such responses has been the goal of R24 (GD3 ganglioside-specific) monoclonal antibody trials, alone and in combination with other agents. This study reports the results of 21 patients treated in a phase IB trial employing R24 (0, 5, 25, 50 mg/m2) administered by continuous i.v. infusion on days 1-5 followed by 3 MU each of interleukin-2 (IL-2) and alpha interferon (alpha-IFN) given subcutaneously on days 8-12, 15-19 and 22-26. R24-related toxicities occurred pre-dominantly at the 25 and 50 mg/m2 doses. One patient (50 mg/m2 R24) exhibited a dose-limiting Grade 4 anaphylaxis. Cytokine-related toxicities required IL-2/alpha-IFN dose reduction in two patients and early termination of treatment in five additional patients. Nine of 20 baseline biopsies showed chronic inflammation; six with lymphocytic tumor infiltration and three where inflammation was confined to the perivascular/peritumoral spaces. No day 8 or 29 biopsies in the R24-treated groups demonstrated treatment-induced tumor lymphocytic infiltrates. However, one patient randomized to no R24 treatment, showed a significant inflammatory tumor lymphocytic infiltration at days 8 and 29. Eighteen of 21 treated patients were evaluable for response. One (5%) patient receiving IL-2/alpha-IFN alone had stable disease lasting 1.5 years. Five (28%) R24, IL-2/alpha-IFN-treated patients had stable disease ranging from 6 to 32 weeks, with one patient remaining alive 2.5 years post-treatment. Although this combined treatment program was generally well tolerated, no objective responses were seen and significant R24-induced tumor lymphocytic infiltrates were not demonstrated.

Cytomegalovirus infection presenting as an apple-core lesion of the colon.

Cytomegalovirus infection is highly prevalent among heart transplant recipients. Symptomatic cytomegalovirus infection can occur in all parts of the gastrointestinal tract. Colonic lesions are usually manifest as hemorrhagic colitis. This is a case of cytomegalovirus colitis presenting as a colonic stricture mimicking a colonic carcinoma. The initial presentation was that of both cellular and humoral rejection with fever, abdominal pain, and microcytic anemia with heme-positive stools. An abdominal computed tomogram was pertinent for a suspicion of carcinoma in the midtransverse colon. After resolution of the rejection episode, colonoscopy was performed, the result of which was abnormal for a short, high-grade stricture in the midtransverse colon. The patient underwent a right hemicolectomy for the suspected tumor. The pathologic specimen showed cytomegalovirus inclusion bodies with acute suppurative ulceration. The early diagnosis and treatment of cytomegalovirus colitis may lead to avoidance of more serious complications such as stricture formation.

Intravesical therapy with vinorelbine tartrate: antitumor activity in orthotopic murine cell carcinoma of the bladder.

OBJECTIVES: To ascertain intravesical vinorelbine tartrate (VNR) antitumor activity against MB-49, a murine transitional cell carcinoma of the bladder (TCC), in an in vivo setting. MATERIALS AND METHODS: C57B1/6J female mice were intravesically implanted with 5 x 10(4) MB-49 cells and treated locally with VNR. Tumor incidence and volume analyses, as well as survival studies were carried out. RESULTS: Tumor incidence was significantly lower in VNR-treated mice (48%, n = 23) than in controls (84%, n = 19), as evaluated sixteen days after MB-49 orthotopic inoculation. Intravesical tumor volume was also significantly smaller in treated mice respect to controls (median [range]: 0.5 [0.4 to 61.8] mm.3 versus 47.7 [4.2 to 179.7] mm.3 respectively, p < 0.001 Kruskal-Wallis test). Median survival duration of the animals treated with VNR was 68 [21 to 68] days, and was significantly greater (p = 0.01, Kruskal-Wallis test) than that of untreated controls (18 [16 to 20] days). CONCLUSION: Intravesical VNR treatment demonstrated an evident antitumor effect against the TCC model assayed. The results obtained suggest a potential use of VNR as intravesical treatment for superficial TCC following transurethral bladder tumor resection to prevent recurrence or retard tumor growth.

Phase I trial of 2B1, a bispecific monoclonal antibody targeting c-erbB-2 and Fc gamma RIII.

2B1 is a bispecific murine monoclonal antibody (BsMAb) with specificity for the c-erbB-2 and Fc gamma RIII extracellular domains. This BsMAb promotes the targeted lysis of malignant cells overexpressing the c-erbB-2 gene product of the HER2/neu proto-oncogene by human natural killer cells and mononuclear phagocytes expressing the Fc gamma RIII A isoform. In a Phase I clinical trial of 2B1, 15 patients with c-erbB-2-overexpressing tumors were treated with 1 h i.v. infusions of 2B1 on days 1, 4, 5, 6, 7, and 8 of a single course of treatment. Three patients were treated with daily doses of 1.0 mg/m2, while six patients each were treated with 2.5 mg/m2 and 5.0 mg/m2, respectively. The principal non-dose-limiting transient toxicities were fevers, rigors, nausea, vomiting, and leukopenia. Thrombocytopenia was dose limiting at the 5.0 mg/m2 dose level in two patients who had received extensive prior myelosuppressive chemotherapy. Murine antibody was detectable in serum following 2B1 administration, and its bispecific binding properties were retained. The pharmacokinetics of this murine antibody were variable and best described by nonlinear kinetics with an average t 1/2 of 20 h. Murine antibody bound extensively to all neutrophils and to a proportion of monocytes and lymphocytes. The initial 2B1 treatment induced more than 100-fold increases in circulating levels of tumor necrosis factor-alpha, interleukin 6, and interleukin 8 and lesser rises in granulocyte-monocyte colony-stimulating factor and IFN-gamma. Brisk human anti-mouse antibody responses were induced in 14 of 15 patients. Several minor clinical responses were observed, with reductions in the thickness of chest wall disease in one patient with disseminated breast cancer. Resolution of pleural effusions and ascites, respectively, were noted in two patients with metastatic colon cancer, and one of two liver metastases resolved in a patient with metastatic colon cancer. Treatment with 2B1 BsMAb has potent immunological consequences. The maximum tolerated dose and Phase II daily dose for patients with extensive prior myelosuppressive chemotherapy was 2.5 mg/m2. Continued dose escalation is required to identify the maximally tolerated dose for patients who have been less heavily pretreated.

Immunohistochemical detection of c-erbB-2 expression by neoplastic human tissue using monospecific and bispecific monoclonal antibodies.

Selected murine monoclonal antibodies (MAb) have been shown to inhibit relevant tumor growth in vitro and in animal models. Recently, bispecific antibodies (BsMAb) have been developed which target cytolytic effector cells via one antibody binding site and tumor antigen by the other specificity. For example, the BsMAb 2B1 possesses specificity for c-erbB-2 and Fc gamma RIII, the low affinity Fc gamma receptor expressed by polymorphonuclear leukocytes (PMN), macrophages and large granular lymphocytes (LGL). The human homologue of the rat neu oncogene, c-erbB-2, has been demonstrated to be amplified in breast, gastrointestinal, lung and ovarian carcinomas. Tumor expression of c-erbB-2 has been shown to be an important prognostic indicator in breast and ovarian carcinomas. The restricted expression of the c-erbB-2 protooncogene product in normal human tissues and the wide distribution of c-erbB-2 expression in such tumors may justify attempts to use an appropriately constructed BsMAb in clinical trials. In this report we have addressed this issue by immunohistochemically evaluating the expression of c-erbB-2 oncogene product in a variety of malignant tumors utilizing 2B1 and the anti-c-erbB-2 monovalent parent of 2B1, 520C9. Among the studied neoplasms, c-erbB-2 expression was detected in 49% of primary carcinomas stained with 520C9 and in 39% of those stained with 2B1. In the group of metastatic tumors, c-erbB-2 oncoprotein was detected in 52% of cases by 520C9 and in 41% by 2B1.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of mutated epidermal growth factor receptor by non-small cell lung carcinomas.

The development of novel immunotherapy strategies for non-small cell lung cancer (NSCLC) will be facilitated by the identification of tumor-specific targets. Although the epidermal growth factor receptor (EGFR) is overexpressed in many cases of NSCLC, its wide distribution in normal tissue may limit its suitability as an immunotherapeutic target. However, mutations within the EGFR that are unique to malignancies may provide specific targets for immunotherapeutic intervention. For example, one mutant form, the type III deletion mutant of the EGFR, that has been identified in glioblastomas contains a novel peptide sequence in its extracellular domain which is detectable by anti-peptide antisera. In this study, the prevalence of this type of mutation of the EGFR in NSCLC was determined. Thirty-two frozen sections of primary NSCLC were examined by immunocytochemistry to determine the presence of native and mutated EGFR. Native EGFR was overexpressed in 12 of the 32 sections and a diffuse cellular distribution of the EGFR type III deletion mutation was identified in five (16%) of the specimens (2 of 13 squamous, 2 of 2 mixed, 0 of 10 adenocarcinoma, and 1 of 7 undifferentiated). This mutated EGFR was not detected in sections of normal breast, lung, skin, ovary, colon, kidney, endometrium, and placenta. The type III EGFR deletion mutant, expressed in some cases of NSCLC, may be a molecularly defined, tumor-specific antigen in lung cancer.

A human tumor xenograft model of therapy with a bispecific monoclonal antibody targeting c-erbB-2 and CD16.

New strategies are required to clinically exploit the ability of monoclonal antibodies to target tumor for lysis by cellular effector mechanisms. In this report we examine the therapeutic effects of 2B1, a bispecific monoclonal antibody with specificity for the extracellular domain of the c-erbB-2 oncogene product and the human Fc gamma receptor, Fc gamma RIII (CD16), describe the characteristics and limitations of this model, and examine the mechanisms underlying the observed responses. The model uses SK-OV-3 human ovarian carcinoma xenografts in scid mice. These cells are susceptible to 2B1-directed lysis by human peripheral blood lymphocytes or lymphokine-activated killer cells, and maintain c-erbB-2 expression in vivo. 125I-labeled 2B1 selectively accumulates in tumor, with a peak of 10.5% injected dose/g of tumor 24 h following its i.v. injection. However, the selectivity of this binding is lessened by 2B1 accumulation in the lungs and other normal organs and persistence in the blood. This is caused by antibody binding to murine lung, colon, stomach, and skin expressing the epitope recognized by the anti-c-erbB-2 component of 2B1 in tumor-bearing, but not normal mice. In treatment studies using various permutations of antibody, human peripheral blood lymphocytes or lymphokine-activated killer cells and interleukin 2, cellular therapy alone had minimal effects on SK-OV-3 xenograft growth, but significantly improved when 2B1 treatment was incorporated. Median survivals increased from 80 +/- 3.5 days with no therapy to 131 +/- 7.3 days following therapy with 100 micrograms 2B1, interleukin 2, and human peripheral blood lymphocytes, with 70% of animals exhibiting no evidence of tumor at day 150. These effects were preserved when the cells were administered in human serum. In contrast, human serum abolished the antitumor effects of 520C9, which is the parent anti-c-erbB-2 antibody of 2B1. Thus 2B1-based therapy has therapeutic effects, without obvious toxicity, despite the targeting of this antibody to normal murine tissues. Since combinations of 2B1 and interleukin 2 may have antitumor properties, mechanisms other than bispecific monoclonal antibody-promoted conjugation of c-erbB-2 antigen-expressing tumor to CD16-expressing effector cells may be involved.

Cytogenetic findings in a breast stromal sarcoma. Application of fluorescence in situ hybridization to characterize the breakpoint regions in an 11;19 translocation.

Cytogenetic analysis of a stromal breast sarcoma revealed a complex karyotype that included a reciprocal 11;19 translocation, along with multiple numerical changes, deletions, and other unbalanced structural rearrangements. Karyotypic abnormalities have not been reported previously in this rare neoplasm that arises from mesenchymal breast tissue, and the t(11;19) is of interest because various types of sarcoma are characterized by specific reciprocal translocations. Because of the pericentric nature of the breakpoints on chromosomes 11 and 19 in the t(11;19), classical cytogenetic banding could not reveal the centromeric origin of the translocation derivatives. Using nonisotopic in situ hybridization with chromosome 11 and 19 alpha-satellite probes, the centromere of each derivative chromosome was determined, and the rearrangement was interpreted as a balanced translocation, t(11;19)(q12 or q13.1;p12 or p13.1). This abnormality has not been described previously in any breast tumor.

Antitumor effects of a bispecific antibody targeting CA19-9 antigen and CD16.

Bispecific murine monoclonal antibodies that target tumor and Fc gamma RIII (CD16) can promote relevant tumor lysis by large granular lymphocytes. For these antibodies to be clinically useful, their properties should be maintained in vivo, where competing human immunoglobulin, shed target antigen, and shed CD16 may be encountered. At a minimum, bispecific antibody antitumor effects should be preserved in whole blood. Furthermore, potentiation of tumor lysis should be reflected by demonstrating the ability of bispecific antibody-retargeted effector cells to infiltrate and mediate lysis of organized tumor. If these characteristics are demonstrated, and there is evidence of in vivo efficacy of bispecific antibody-based therapy in a relevant animal model, further clinical development of such antibodies would be warranted. In this report the ability of CL158 bispecific antibody supernatants to mediate lysis of SW948 tumor growing in monolayer is shown to be preserved in the presence of interleukin 2-activated whole blood. When SW948 cells were grown in vitro as multicellular human tumor spheroids, incubation with interleukin 2-activated lymphocytes (LAK cells) and CL158 led to structural and widespread necrosis. This was dependent on CL158 and resistant to competition by pooled human immunoglobulin or interleukin 2-exposed whole blood. These effects were not promoted by the monospecific antibodies produced by the parent clones of CL158 and were not observed when the IgG2a variant of CA19-9 antibody, which mediates conventional antibody-dependent cellular cytotoxicity, was used instead of its bispecific derivative. To examine the efficacy of bispecific antibody-based treatments on in vivo tumor, scid mice bearing early s.c. SW948 xenografts were treated with interleukin 2 for 5 consecutive days, supplemented by three i.v. injections of 10(7) human LAK cells and various antibodies. Treatment of mice bearing SW948 tumors with LAK cells did not retard tumor growth, but when CL158 was added, significant delays in tumor growth were observed. Tumor growth delay required treatment with both LAK cells and the bispecific antibody. Treatment with the IgG2a variant of CA19-9 antibody, alone or with LAK cells, had no effects on tumor growth. Although the mechanisms of these antitumor effects require further study, it is clear that human LAK cell treatment of animals bearing early, established s.c. tumors is enhanced by the addition of bispecific antibodies with relevant binding characteristics. When compared with the IgG2a isotype variant of CA19-9 monoclonal antibody, this bispecific antibody offers the advantages of preservation of activity in physiological conditions, infiltration and disruption of organized tumor in vitro, and antitumor effects in a relevant xenograft model.(ABSTRACT TRUNCATED AT 400 WORDS)

A tobacco-specific N-nitrosamine or cigarette smoke condensate causes neoplastic transformation of xenotransplanted human bronchial epithelial cells.

Using a xenotransplantation system in which immortalized nontumorigenic human bronchial epithelial cells (BEAS-2B cells) are grown in deepithelialized rat tracheas that are subcutaneously transplanted into athymic nude mice, we exposed BEAS-2B cells either to cigarette smoke condensate or to the tobacco-specific N-nitrosamine 4-(methylnitrosamine)-1-(3-pyridyl)-1- butanone. After 6 mo the carcinogen-exposed BEAS-2B cells were neoplastically transformed to invasive adenocarcinomas. Cell lines obtained from xenografts exposed in vivo to chemicals exhibited several features typical of malignant lung cancer cells, such as increased in vivo invasiveness that correlated well with enhanced type IV collagenolytic activity, resistance to serum-induced growth inhibition, and increased expression of transforming growth factor alpha and its cellular-membrane receptor. Invasiveness, similar to that seen after exposure to phorbol esters, was also detected after in vitro exposure of BEAS-2B cells to cigarette smoke condensate. Collectively, these data indicate that cigarette smoke condensate and N-nitrosamine 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone induce in vivo phenotypic changes in BEAS-2B cells similar to the progressive changes that occur during human lung carcinogenesis.

Bispecific monoclonal antibody regulation of Fc gamma RIII-directed tumor cytotoxicity by large granular lymphocytes.

Bispecific monoclonal antibodies (BsMAbs) prepared by somatic cell fusion bind monovalently to their targets and yet are extremely potent enhancers of target cell lysis by relevant effector cells. The mechanisms underlying this efficiency are not known. To investigate this property, we studied the ability of selected antibodies to modulate potentiation of tumor lysis by a bispecific antibody (CL158) which targets Fc gamma RIII-expressing cells, via the 3G8 epitope, to malignant cells expressing CA19-9 antigen. Antibodies directed against the 3G8 and B73.1 epitopes of Fc gamma RIII efficiently inhibited BsMAb-mediated SW948 tumor cell lysis by interleukin-2 (IL-2)-activated lymphocytes (PBLs). Unexpectedly, Leu 19 antibody reversed antibody-dependent but not antibody-independent lysis of 51Cr-labeled SW948 cells by IL-2-activated PBLs in a concentration-dependent fashion. Leu 19 binds to CD56, a neural cell adhesion molecule (N-CAM) isoform expressed by large granular lymphocytes (LGLs). The effects of Leu 19 on bispecific antibody promotion of lysis were due to competition for binding to the 3G8 epitope of Fc gamma RIII and led to inhibition of binding between LGLs and SW948 cells. Leu 19 did not inhibit antibody-dependent lysis by the monospecific, bivalent IgG2a variant of CA19-9 antibody. These studies show that competition assays can be useful in dissecting the relevant mechanisms underlying BsMAb-promoted lysis. Steric constraints between effector cell trigger molecules (i.e., Fc gamma RIII) and CAM such as N-CAM may regulate the function of these molecules. Understanding the roles of diverse CAM in this phenomenon will facilitate efforts to expand and use defined effector cell populations with maximal lytic potential and to identify potentially responsive tumor phenotypes.

Potentiation of tumor lysis by a bispecific antibody that binds to CA19-9 antigen and the Fc gamma receptor expressed by human large granular lymphocytes.

Murine monoclonal antibody therapy of human cancer rarely induces clinical responses. Antibody-induced cellular infiltrates rarely accumulate at sites of tumor, even in clinically responding lesions. Thus, the ability of these antibodies to promote host effector cell-mediated lysis of tumor via antibody-dependent cellular cytotoxicity (ADCC) has not been harnessed by existing treatment approaches. One potential explanation is that ADCC requires binding of antibody Fc domains to cellular Fc gamma receptors, and therapeutically administered murine antibodies must compete with vast excesses of human IgG for Fc gamma receptor occupancy. Chemically linked antibody heteroconjugates that bind selected target and effector cell structures via distinct Fab portions can mediate lysis of malignant cells in vitro in the presence of human serum. This approach addresses a potentially major obstacle to antibody therapy. Production of bispecific monoclonal antibodies with similar specificities and superior in vivo biodistribution characteristics would thus have potential clinical applications. We have prepared and purified a bispecific, monovalent monoclonal antibody and evaluated its in vitro effects. The IgG1-secreting hybridoma line 3G8 (alpha-human Fc gamma R III) was fused with the hybridoma line CA19-9, which produces an IgG1 antibody that binds to a glycoprotein shed by gastrointestinal cancers. Multiple clones with bispecific binding properties were identified. CA19-9 x 3G8 clonal supernatants and purified antibody, but not the parent antibodies, efficiently mediated specific in vitro lysis of cells of the SW948 line by human large granular lymphocytes (LGLs). Human serum-resistant target cell lysis augmentation at low effector:target ratios was seen using picogram amounts of antibody. In contrast, the IgG2 alpha variant of CA19-9, which also promotes ADCC by LGLs, was unable to augment lysis of SW948 cells when effectors were preincubated with human serum. This bispecific, monovalent monoclonal antibody is an efficient promoter of the anti-tumor effects of LGLs in physiological concentrations of human serum. In vivo models that evaluate treatment efficacy and promotion of inflammatory tumor infiltrates by bispecific monoclonal antibodies are required to assess the therapeutic potential of these novel constructs.