Role of alloantigens in natural killing. Allogeneic but not autologous tumor biopsy cells are sensitive for interferon-induced cytotoxicity of human blood lymphcoytes.

Blood lymphocytes of patients with solid tumors were assayed for cytotoxicity against autologous and allogeneic primary tumor cells. The lymphocytes killed autologous tumor cells in 7 of 25 cases (28%) and allogeneic tumor cells in 2 of 37 tests (5%). Lymphocytes from healthy donors were rarely cytotoxic for the biopsy cells, which indicates that these cells have low natural kill sensitivity. The autoreactivity that may reflect the immunological recognition of tumor cells was not altered by pretreatment of the effectors with interferon (IF). In contrast, killing of allogeneic tumor biopsy cells was induced by IF in approximately 50% of tests, with the lymphocytes of both the tumor patients and the healthy donors. The mechanism of the alloreactivity is most likely a consequence of IF-induced polyclonal activation of cytotoxic potential and the lymphocytes that are committed to recognize the alloantigens expressed on the particular target manifest the killing function. When the biopsy cells were explanted and kept in culture for 5-6 d, their susceptibility for the lymphocyte damage increased, and they were killed by the IF-treated cells also in autologous combinations. Whether this change in sensitivity is a result of qualitative or quantitative changes in antigen expression or of other changes in the properties of the cell membrane is unknown.

Advanced statistical techniques applied to comprehensive FTIR spectra on human colonic tissues.

PURPOSE: Colon cancer is a major public health problem due to its high disease rate and death toll worldwide. The use of FTIR microscopy in the field of cancer diagnosis has become attractive over the past 20 years. In the present study, the authors investigated the potential of FTIR microscopy to define spectral changes among normal, polyp, and cancer human colonic biopsied tissues. METHODS: A large database of FTIR microscopic spectra was compiled from 230 human colonic biopsies. The database was divided into five subgroups: Normal, cancerous tissues, and three stages of benign colonic polyps, namely, mild, moderate, and severe polyps, which are precursors of carcinoma. All biopsied tissue sections were classified concurrently by an expert pathologist. The authors applied the principal components analysis (PCA) model to reduce the dimension of the original data size to 13 principal components. RESULTS: While PCA analysis shows only partial success in distinguishing among cancer, polyp, and the normal tissues, multivariate analysis (e.g., LDA) shows a promising distinction even within the polyp subgroups. CONCLUSIONS: Good classification accuracy among normal, polyp, and cancer groups was achieved with a success rate of approximately 85%. These results strongly support the potential of developing FTIR microscopy as a simple, reagent-free tool for early detection of colon cancer and, in particular, for discriminating among the benign premalignant colonic polyps having increasing degrees of dysplasia severity (mild, moderate, and severe).

Can Fourier transform infrared spectroscopy at higher wavenumbers (mid IR) shed light on biomarkers for carcinogenesis in tissues?

Fourier transform infrared microspectroscopy (FTIR-MSP) has shown promise as a technique for detection of abnormal cell proliferation and premalignant conditions. In the present study, we investigate the absorbance in the sensitive wavenumber region between 2800 and 3000 cm(-1), which has been known to be due to the antisymmetric and symmetric stretching vibrations of CH2 and CH3 groups of proteins and lipids. We report common biomarkers from this region that distinguish between normal and malignant tissues and cell lines. Based on our findings, we propose that the wavenumber region around 2800 to 3000 cm(-1) in the FTIR spectra of cells and tissues could provide valuable scientific evidence at the onset of premalignancy and may be used for ex vivo and in vitro detection of carcinogenesis. To further examine the utility of these markers in cancer diagnosis and management, they are tested successfully in monitoring the changes occurring in leukemia patients during chemotherapy.

Heterogeneity of human natural killer (NK) cells: enrichment of NK by negative-selection with the lectin from Erythrina cristagalli.

A novel technique for the isolation and enrichment of human natural killer (NK) cells from peripheral blood mononuclear cells (MNC) is described. Negative selection of MNC with the lectin from Erythrina cristagalli (ECA), whether by panning or agglutination in solution, resulted in a population of lymphocytes (5-20% of original MNC) highly enriched in cells exhibiting NK function. This enrichment was evident by a significant increase (range of 3-50-fold) in cells with large granular lymphocyte (LGL) morphology, K562 tumor-binding cells, cytotoxic activity, and cells expressing NK phenotypic markers (Leu 11+, OKM1+). Analysis of the cytolytic specificity of the cells demonstrated that the lytic spectrum was typical of endogenous NK. The effector cells were responsive to augmentation of cytotoxic potential by lymphokines (IL-2, IFN alpha, and IFN gamma) and capable of antibody-dependent cell-mediated cytotoxicity (ADCC). ECA-negative [ECA(-)] cells were equivalent to NK isolated by Percoll gradient fractionation. NK heterogeneity was demonstrated by the observation of a small percentage (1-5% of MNC) of NK in the ECA(+) population. This technique was found to be advantageous for the study of NK heterogeneity and NK biology.

Postsplenectomy pericardial effusion in two patients with myeloid metaplasia.

Self-limited sympathetic pleural effusion is a well-known nonspecific sequela following any upper abdominal surgery, including splenectomy. However, to our knowledge, there is no report in the literature on sympathetic pericardial effusion immediately following splenectomy. We describe two patients with agnogenic myeloid metaplasia and refractory hemolytic anemia in whom pericardial effusion appeared a few days after splenectomy. The possible pathophysiologic mechanism is discussed.

IFN-alpha-induced modulations of the events in human mixed lymphocyte cultures.

Autologous mixed lymphocyte cultures (AMC) with T-enriched subset of human blood lymphocytes as responders and B-cells or plastic adherent cells as stimulators and allogeneic mixed lymphocyte cultures (MLC) were assayed for blastogenesis and generation of cytotoxic potential. The activated cells lyzed K562 and Daudi, autologous and allogeneic PHA-blasts. The AMC population affected the autologous and allogeneic blasts at a similar strength and there was no indication for selective effects. B-Blasts induced with Staphylococcus aureus were not lyzed. The MLC populations had a stimulation-specific cytotoxic component. This was revealed by the stronger effect against the stimulator PHA-blasts and by the lysis of the stimulator B-blasts. Short-time interferon (IFN) treatment prior to the lytic assay enhanced the anti-Daudi and anti-K562 lytic activity of the AMC and MLC populations. With AMC the lytic efficiency against the autologous and allogeneic PHA-blasts were not changed while with MLC they were also elevated. This increase was confined to the non-specific component of the cytoxicity. The proliferation of lymphocytes was suppressed when interferon was added at the initiation of the mixed cultures. On a per-cell basis the cytotoxic potential of these cultures were stronger. In the MLC the stimulation-specific component increased more substantially than the effect against the non-specific targets. It is possible that the IFN-induced modification of the culture conditions such as suppression of the initial proliferation favored the growth of the specific clone. Re-exposure of these cells to another dose of interferon prior to the lytic assay had no effect on the lytic potential.

A human monoclonal anti-DNA antibody derived from a patient with polymyositis having the common lupus 16/6 idiotype.

A human IgM monoclonal antibody (Pol-1, SA-1) was generated by the human hybridoma technique from the peripheral blood lymphocytes (PBL) of a patient with active polymyositis. The antibody was found to bind to ssDNA, dsDNA, poly(I) and poly(G) and to carry the common lupus anti-DNA antibody idiotype (16/6 Id). Another human IgM monoclonal antibody (Pol-2, SA-2) produced by similar methods from the PBL of the same patient while in remission lacked the ligand-binding capacities of Pol-1 SA-1 and did not have the 16/6 Id. Analyses of 19 sera samples from patients with polymyositis showed no antinuclear antibodies, excluding a 40% prevalence of the 16/6 Id. The serum of the patient whose lymphocytes were employed to generate the hybridoma was negative for anti-DNA activity as well as for the 16/6 Id. This study suggests that the hybridoma technique may enable expression of dormant idiotypic affinities which do not normally appear in sera.

Naturally-occurring tumor-reactive autoantibodies: a monoclonal antibody from normal mice reacts with tumor cells and with DNA.

A natural IgM monoclonal antibody, 1.67, was generated from apparently healthy unstimulated BALB/c mice. This antibody reacted with L5178Y murine T cell lymphoma, with human Raji cells, and with several normal cells. Further analysis of its ligand binding capacity disclosed strong binding to single-stranded DNA (ssDNA). However, this naturally-occurring monoclonal antibody binds to different epitopes on cell membranes and on DNA than another anti-DNA monoclonal antibody (18/103/1) from human origin. This conclusion was based on competition assays. Furthermore, NOA 1.67 lacks the 16/6 idiotype expressed on the 18/103/1 antibody. The 16/6 idiotype is shared by human and mouse lupus monoclonal autoantibodies that bind simultaneously to lymphoid cells and DNA. This is a first report on a natural autoantibody that binds to malignant and to normal cell membrane(s) as well as to ssDNA. It may have regulatory functions controlling malignancy and or autoimmunity.

Autoantibodies in male homosexuals and HIV infection.

We have used ELISA to study the frequency of autoantibodies to several antigens in the serum of 17 male homosexuals (MHS) negative for HIV (HIV-), 11 asymptomatic HIV seropositive MHS (HIV+) and patients with ARC (N = 15) or AIDS (N = 13), and compared them to 20 matched healthy heterosexual controls. Serum antibody binding to histones, cardiolipin, ss-A, ss-B and Sm was found to be significantly higher in each of the MHS groups studied as compared to controls (P less than 0.001), and was also increased in the HIV+ patients vs. the HIV- group (P less than 0.05). In contrast, no increase in autoantibodies to ss-DNA, ds-DNA, poly(I), poly(G) or RNP were found in any of the groups tested. These results enlarge the spectrum of autoimmunity previously reported in HIV infection and identify a similar pattern to a lesser degree, already present in HIV- MHS, suggesting a role for HIV or concomitant virus infections in its pathogenesis.

Cation requirement of natural, in-vitro generated and antibody dependent killing exerted by human lymphocytes.

Three cytotoxic systems, NK, ADCC and the seemingly indiscriminative cytotoxicity of in vitro activated lymphocytes (cultivated with K 562) were found to require Ca++ and did not function in Mg++. The optimum concentration of Ca++ was identical for the three systems and for various lymphocyte subsets. 15 minutes after initiation of the interaction cell damage had already occurred in all three systems.

Cytolysis of actinomycin D-treated target cells by cell-free supernatants from human monocytes.

The lytic mechanism of human peripheral blood monocytes was studied by using as targets actinomycin D-treated WEHI-164, an NK-insensitive murine fibrosarcoma cell line. Monocytes, but not lymphocytes, lysed WEHI-164 target cells pre-treated with actinomycin D within 6 h in 51Cr-release assays. Because cytolysis could not be inhibited competitively by unlabeled WEHI/D target cells, contact-independent mechanisms of cytolysis were investigated. Cell-free supernatants collected from monocytes cultured for 4-6 h at 37 degrees C lysed target cells as effectively as effector cell preparations of monocytes. Supernatants from lymphocytes cultured in parallel were not cytolytic. Cytolytic activity was not detected in supernatants from preparations of monocytes that were held on ice. However, monocytes produced cytolytic activity whether they were isolated by adherence or remained unseparated in suspensions of mononuclear cells. The cytolytic activity in cell-free supernatants (CFS) from monocytes was unaffected by incubation with protease inhibitors. CFS activity was destroyed by heat. Storage of CFS at 37 degrees, 22 degrees, 4 degrees, or -20 degrees C for 24 h decreased cytolytic activity; however, loss of cytotoxicity was minimized by storage at 4 degrees C. The cytolytic substance detected in 4-h CFS from monocytes appeared to be a protein(s) based on the sensitivity of the cytolytic activity to proteases. Cytolytic activity of CFS eluted from Sephacryl 200 in a single peak with an apparent molecular weight between 25,000 and 45,000 daltons.

Crohn's disease isolated to the appendix: truths and fallacies.

Twenty-five cases of Crohn's disease confined to the appendix were reported in eight hospitals in Israel during a 15-year period. Review of the histologic slides confirmed the diagnosis in 22 cases. Re-evaluation of these 22 patients included physical examination and radiologic studies of the small and large bowel. Rectosigmoidoscopy was performed in 16 patients. Signs and symptoms of Crohn's disease at other sites in the gastrointestinal tract did not occur during follow-up periods of two to 15 years (mean, 6.4 years) after appendectomy. This study and a review of the literature indicate that in most cases (93 per cent) Crohn's disease initially limited to the appendix is not a predictor of subsequent involvement of another portion of the bowel. It is concluded that the so-called Crohn's disease isolated to the appendix is a form of chronic granulomatous and follicular appendicitis of unknown etiology that is unrelated nosologically in the majority of the cases to Crohn's disease proper.

Differential expression of renal growth hormone receptor and its binding protein in experimental diabetes mellitus.

Growth hormone (GH) may have a role in the development of diabetic nephropathy. The effect of experimental diabetes on renal expression of the growth hormone receptor gene products, including the receptor itself (GHR) and its binding protein (GHBP) was examined. Adult female rats received i.v. streptozotocin and were killed at 7, 30, 90 and 180 days after the induction of diabetes. Diabetic animals had a pronounced increase in kidney weight and progressive albuminuria. In renal cortex, no change was seen in GHR mRNA levels throughout the observation period of 6 months, while a significant increase in cortical GHBP mRNA levels was observed after 1 month of diabetes and sustained for the rest of the study period. Immunohistochemical analysis of kidney sections revealed a stronger staining for GHBP at the cortical and inner medullary areas in the diabetic animals. These data indicate that although the GHR and GHBP mRNAs originate from the same gene, their renal levels are differentially regulated during the development of experimental diabetic kidney disease, suggesting a functional role for GHBP.

Acute blast crisis with EBV-infected blasts, in a patient with chronic myeloid leukemia, and vasculitis.

Unless they undergo transplantation, all patients with chronic myeloid leukemia (CML) will eventually develop a late phase of acute blast crisis (ABC). Although additional chromosomal abnormalities to the Philadelphia (Ph) chromosome may herald ABC in many CML cases, the mechanisms leading to this fatal event are obscure. Viral etiology, including the Epstein-Barr virus (EBV) has never been implicated in the pathogenesis of ABC in CML. Iloprost is an analogue of epoprostenol (prostacyclin; PGI2) commonly used for the treatment of peripheral vascular diseases and acts via inhibition of platelet activation, and by vasodilation. A case of ABC with blasts of undetermined lineage showing EBV infection in a male patient with Ph positive CML is described here. This unusual event developed during a course of treatment with the prostacyclin analogue, iloprost administered for vasculopathic leg ulcers. The proliferating blasts stained positively by immunohistochemistry only for the leukocyte common antigen (LCA/CD-45), and the EBV-latent membrane protein 1 (LMP-1). The only chromosomal abnormality detected by cytogenetic analysis was the conventional Ph-chromosome. It is suggested that ABC in this case of CML, was associated with EBV-activated blasts of undetermined lineage.

Fourier transform infrared microspectroscopy as a quantitative diagnostic tool for assignment of premalignancy grading in cervical neoplasia.

The early diagnosis and proper identification of cervical squamous intraepithelial lesions plays an important role in a good prognosis for the patient. However, the present practice of screening based on PAP (Papanicolaou) smear and histopathology makes it tedious and prone to human errors. We assess the validity of FTIR microspectroscopy (FTIR-MSP) of biopsies as a method to properly assign the correct stage of premalignancy in patients with symptoms of cervical intraepithelial neoplasia. For the first time we evaluate the biopsies based on the FTIR spectra for different grades of neoplasia in tandem with probabilistic neural networks (PNNs) and histopathology. The results show that the grading of neoplasia based on FTIR-MSP and a PNN differentiates the normal from premalignant with a high level of accuracy. The false positive identification of the normal as cervical intraepithelial neoplasia 1 (CIN1), CIN2, and CIN3 patients is 9.04, 0.01, and 0.01%, respectively. The false negative identification of CIN2 patients as normal and CIN1 patients is 0.01 and 4.4%, respectively. Similarly, the false negative identification of CIN3 patients as normal, CIN1, and CIN2 is 0.14, 6.99, and 9.61%, respectively. The small errors encountered in the grading are comparable to current methods, encouraging advanced studies for the development of mechanized equipment for the diagnosis and grading of premalignant cervical neoplasia.

Characteristic absorbance of nucleic acids in the Mid-IR region as possible common biomarkers for diagnosis of malignancy.

FTIR spectroscopy has been extensively used to understand the differences between normal and malignant cells and tissues. In the present study, FTIR microspectroscopy was performed on biopsies to evaluate parameters deduced from changes in nucleic acid absorbance monitored at various characteristic wavenumbers in the Mid-IR region. The data showed that there were differences in the spectra of normal and malignant tissues from several organs such as colon, cervix, skin and blood with respect to absorbance due to nucleic acids. Similar results were observed in the case of cell lines that were transformed to induce carcinogenesis. Of the several ratios examined for consistency in differentiating cancer and normal tissues, the I(996 cm(-1))/I(966 cm(-1)) showed promise as a distinguishing parameter and was comparable to the I(1121 cm(-1))/I(1020 cm(-1)) ratio reported in many earlier studies. The absorbance of nucleic acids is presented with an emphasis on the application of FTIR microspectroscopy for diagnosis of malignancy. Our results indicate that usage of nucleic acid absorbance yield statistically significant parameters, which could differentiate normal and cancerous tissues.

Open lateral sphincterotomy is still the best treatment for chronic anal fissure.

Chronic anal fissure is the main cause of anal pain and is very common. Many treatment modalities have been tried-none is ideal. Recently, topical nitric oxide releasing agents and botulin toxin injections have been used, trying to replace surgery. No long-term experience is available. Personal experience with 2,108 open, ambulatory, lateral sphincterotomy with follow-up of 4-20 years is presented.

Is routine crossmatching for two units of blood necessary in elective surgery?

The usual preparation for many elective procedures calls for typing and crossmatching for 2 units of blood. Most of the blood prepared is not used. Every unit of blood kept for a specific patient is removed from the blood bank pool, thereby reducing its resources. Fourteen hundred of the most frequent elective operations were examined with regard to their blood utilization. A transfusion index was developed to determine whether preoperative crossmatching was justified. Applying the index to 1,400 elective procedures, it was found that only vascular procedures justified preoperative crossmatching. The safety of transfusion (elective and emergency) based on type and antibody screening is explained. The constant increase in blood consumption (as for open heart surgery) and the small increase in blood donations has created a blood shortage. It is suggested that surgeons stop requesting crossmatching for 2 units for those elective procedures in which blood is rarely used. This would decrease the workload in the blood bank and increase the blood bank pool without compromising patients' safety.

Is routine use of the nasogastric tube justified in upper abdominal surgery?

Three hundred patients who underwent upper abdominal operations were studied. One hundred fifty patients were treated by insertion of a nasogastric tube, and 150 patients were treated without it. The incidence of postoperative pneumonia was 10 times higher in the patients treated with a nasogastric tube. Pneumonia was directly related to the patient's age and the duration of the tube's use. In view of the disadvantages and complications of the nasogastric tube, its routine use appears unjustified. It should be reserved as a tool for treating postoperative complications such as paralytic ileus and acute gastric dilatation.

The major role of the operative cholangiogram within the indications for common bile duct exploration.

The accuracy of the clinical indications for common bile duct exploration was studied in a group of 495 patients. The patients were divided into two groups: operative cholangiography was performed in 5 per cent of cases in Group A and 62 per cent of cases in Group B. The results demonstrate the high efficiency of this procedure, its safety, accuracy (82%), and its effectiveness in the discovery of unexpected stones and in shortening of hospitalization. Analysis of the results point out the indications for intraoperative cholangiography: It should be carried out in all biliary tract operations, except for those in which other clinical indications for exploration already exist (palpable stones in the common bile duct or the combination of dilated common bile duct either with jaundice at the time of operation or with history of severe jaundice in the past). Under all other circumstances an operative cholangiogram should be performed.