Selecting antibodies to detect HER2 overexpression by immunohistochemistry in invasive mammary carcinomas.

There is an increasing clinical demand for HER2 analysis in breast cancer, especially since the release of trastuzumab. The authors assessed the ability of immunohistochemistry to detect HER2 overexpression in invasive mammary carcinomas (IMC) using five antibodies. Paraffin-embedded samples of 86 IMCs (T2N0) were used to compare the immunohistochemical overexpression of HER2 using two polyclonal antibodies (HercepTest [DAKO] and A0485 [DAKO]) and three monoclonal antibodies (CB11 from two different laboratories, Biogenex and Novocastra, and 4D5 [Genentech]). All immunostainings were scored according to the FDA-approved HercepTest recommendations. The HercepTest-positive cases were compared with gene amplification by FISH (Oncor Inform, Ventana). The HercepTest was positive in 31 of the 86 cases (36.1%). The DAKO antibody A0485 was positive in 25 of the 66 (37.8%). Monoclonal antibody 4D5 was positive in only 15 of the 86 cases (17.4%). There was almost total agreement in results between the two CB11 antibodies: 25 of the 86 positive cases (29.1%). All cases positive for CB11 or 4D5 were HercepTest positive. Most of the HercepTest 2+ cases were negative when using either monoclonal antibody. FISH was positive in 19 of the 20 HercepTest 3+ cases and negative in 5 HercepTest 2+ cases. Three CB11-2+ cases showed no amplification by FISH. In three FISH-positive cases the immunohistochemistry showed no overexpression by all antibodies used. These findings suggest that immunohistochemistry may be used reliably as a primary methodology for evaluating HER2; however, the use of polyclonal antibodies may not be adequate to assess HER2 overexpression. CB11, regardless of the manufacturer (Biogenex or Novocastra), showed better concordance with FISH (kappa=0.83) than did the polyclonal antibodies.     

Catheter drainage of symptomatic postoperative mediastinal effusion guided by computed tomography. A nonoperative approach

Postoperative mediastinal effusion after cardiac operations is a common phenomenon and has a potential for serious complications, such as tamponade necessitating urgent drainage. Computed tomography of the chest provides excellent postoperative visualization of the mediastinum. Catheter insertion or paracentesis guided by computed tomography has been used to accomplish nonoperative drainage of symptomatic postoperative mediastinal effusion in six cases. This technique offers simplicity, safety, and cost effectiveness.     

Effects of trifluoperazine, ouabain, and ethacrynic acid on intestinal calcium transport

Bidirectional steady-state calcium fluxes were measured in vitro under short-circuited conditions in segments of rat duodenum and descending colon. The calcium-activated ATPase (Ca-ATPase) inhibitor trifluoperazine (TFP, 0.1 mM) reduced net calcium absorption in both tissues by decreasing the absorptive flux from mucosa to serosa (Jm leads to s) without consistently altering the secretory flux from serosa to mucosa. 1,25-Dihydroxyvitamin D3 administration (50 ng/day for 4 days) increased net calcium absorption by increasing Jm leads to s, and TFP reduced Jm leads to s to the same extent across tissues from vehicle- or 1,25-dihydroxyvitamin D3-treated animals. Na-K-ATPase inhibitors ouabain and ethacrynic acid both reduced short-circuit current without affecting calcium fluxes. These data suggest that Ca-ATPase, located in the basolateral membrane of intestinal epithelial cells, plays a role in the transepithelial transport of calcium. More general effects of TFP on intestinal epithelium may also contribute to the reduction in calcium fluxes. Duodenal and descending colon calcium transport appears independent of transcellular sodium transport mediated by Na-K-ATPase.     

Intravenous teicoplanin does not prevent Clostridium difficile associated diarrhea

A 59-year-old man with the diagnosis of endocarditis of the mitral valve due to Streptococcus mitis was treated with penicillin G, gentamicin, and later with clindamycin as inpatient for 3 weeks. Thereafter outpatient therapy with parenteral teicoplanin 3 × per week was initiated. After 17 days of teicoplanin treatment he developed severe diarrhea, and stool samples were positive for Clostridium difficile toxin. In addition to the ongoing parenteral therapy with teicoplanin, oral teicoplanin was administered. On the third day of this regimen the diarrhea and other disabling symptoms subsided, and test results for C. difficile toxin became negative. Oral teicoplanin was continued for 10 days and cleared C. difficile effectively after treatment as assessed by consecutive stool cultures (until 60 days thereafter). The parenteral administration of teicoplanin could not prevent the onset of C. difficile associated diarrhea in this patient, who previously had been treated with clindamycin. Thus, the administration of parenteral teicoplanin does not seem to be a treatment option for C. difficile associated diarrhea in patients in which oral therapy is not possible.     

A sensitive bioassay for chicken interleukin-18 based on the inducible release of preformed interferon-gamma

Conventional tests for measuring the biological activity of chicken interleukin (IL)-18 require primary chicken spleen cells. We now describe a sensitive bioassay that is based on interleukin-18-induced release of interferon (IFN)-gamma by a permanent chicken cell line. In B19-2D8 cells, cytoplasmically stored interferon-gamma is quickly secreted in response to interleukin-18 exposure.     

The down-regulation of FcgammaRII and FcgammaRIIIB by N-formyl-methionyl-leucyl-phenylalanine (FMLP) depends on secretory events in human neutrophils

We have previously demonstrated that N-formyl-methionyl-leucyl-phenylalanine (FMLP) induces down-regulation of FcgammaRs on human neutrophils (PMN) modifying different FcgammaR-dependent functions. The aim of this work was to assess the cellular mechanisms by which FMLP exerts this effect on FcgammaRs. The role of the microfilament and cytoskeletal apparatus in this process was evaluated using cytochalasin B (CB), an inhibitor of microfilament functions. The expression of FcgammaRIIIB and FcgammaRII after CB + FMLP treatment was drastically diminished when compared to FMLP-treated cells. Neutrophil degranulation induced by FMLP affect only 22% of the cells in response to FMLP. However, the FcgammaRs of the whole PMN population were reduced, suggesting that secretory products could be responsible for the down-regulation induced by FMLP or FMLP + CB. In fact, supernatants from FMLP-treated PMN also induced FcyRs down-regulation on naive neutrophils. Moreover, supernatants from FMLP + CB-treated PMNs exerted a higher effect. Data obtained from permeabilized PMN show that after FMLP treatment there is an intracellular depletion of both FcgammaRIIIB and FcgammaRII. In addition, the FcgammaR down-regulation is abrogated by phenyl methyl sulfonyl fluoride (PMSF) but not by other protease inhibitors such as pepstatin, thiorphan, phosphoramidon and leupeptin, suggesting a role for serine protease(s) in this process.     

Metaplastic spindle cell breast tumors arising within papillomas, complex sclerosing lesions, and nipple adenomas.

Micropapillomas/papillomas and complex sclerosing lesions of the breast have been associated with a slightly increased risk for subsequent carcinoma, although benign squamous metaplasia and reactive hypercellular stroma are seen within these lesions. There are few reports of these fibrosclerotic lesions associated with metaplastic tumors. Here we describe a series of metaplastic tumors arising within fibrosclerotic breast lesions. Thirty-three metaplastic tumors associated with fibrosclerotic lesions were selected from a breast pathology consultative practice. Relevant clinical and pathological features were reviewed. Representative sections were evaluated immunohistochemically for expression of cytokeratins, vimentin, and smooth muscle and muscle-specific actins. Both the metaplastic component (spindled and squamous cells) and the glandular elements were graded. The metaplastic tumors arose within papillomas (20 cases), complex sclerosing lesions (7 cases), both papilloma and complex sclerosing lesions (3 cases), and nipple adenoma (3 cases). A majority of the metaplastic tumors showed a dominant spindle cell component with various degrees of atypia, ranging from fibromatosis-like (16 cases) to low-grade (13 cases), intermediate-grade (2 cases), and high-grade (2 cases) fibrosarcoma phenotype. Squamous metaplasia was present in 25 cases, and low-grade glandular elements, in 21 cases. Eleven tumors had a low-grade adenosquamous growth pattern. Ductal carcinoma in situ was present in 7 cases, and invasive mammary carcinoma, in 5 cases. The very low-grade tumors were histologically similar to limited areas of stromal reaction and myofibroblastic proliferation, seen in partially sclerotic micropapillomas/papillomas and complex sclerosing lesions, but usually more cellular. Cytokeratin positivity (13+/13 tested) supports the metaplastic nature of the more plump spindled cells. The spindle cells were also positive for vimentin (8+/8 tested) and smooth muscle (2+/5 tested) and muscle-specific actins (6+/6 tested). Spindle cell metaplastic tumors, from fibromatosis-like to fibrosarcoma, may arise within a variety of fibrosclerotic breast lesions.     

Cellular Immunity in COVID-19 Convalescents with PCR-Confirmed Infection but with Undetectable SARS-CoV-2–Specific IgG

We investigated immune responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among a group of convalescent, potential blood donors in Germany who had PCR-confirmed SARS-CoV-2 infection. Sixty days after onset of symptoms, 13/78 (17%) study participants had borderline or negative results to an ELISA detecting IgG against the S1 protein of SARS-CoV-2. We analyzed participants with PCR-confirmed infection who had strong antibody responses (ratio >3) as positive controls and participants without symptoms of SARS-CoV-2 infection and without household contact with infected patients as negative controls. Using interferon-γ ELISpot, we observed that 78% of PCR-positive volunteers with undetectable antibodies showed T cell immunity against SARS-CoV-2. We observed a similar frequency (80%) of T-cell immunity in convalescent donors with strong antibody responses but did not detect immunity in negative controls. We concluded that, in convalescent patients with undetectable SARS-CoV-2 IgG, immunity may be mediated through T cells.