Jacob J. Steinberg

Morphologic pattern of tenascin as a diagnostic biomarker in colon cancer

Immunohistochemical methods were used to study the pattern of expression of tenascin (TN) in invasive colon cancer and its relation to prognosis.

Contrasting effects of identical nutrients given parenterally or enterally after 70% hepatectomy

Based on clinical observations, we hypothesized that prolonged parenteral nutrition (in contrast to enteral nutrition) is detrimental after major hepatic resection. Male Sprague-Dawley rats (300 to 380 g) anesthetized with intraperitoneal sodium pentobarbital had 70% hepatic resection and jugular vein and gastrostomy catheterizations using aseptic techniques and were divided randomly into three groups: (1) total parenteral nutrition (TPN) (nutrients via central vein), (2) total enteral nutrition (TEN) (identical nutrients via gastrostomy), and (3) standard oral feeding (SOF) (chow and water ad libitum). Unused catheters were plugged. In the first set of experiments (n = 42), nutrient intake was formulated to approximate the nutritional intake of normal rats, 216 kcal/kg/d. Infusate was 15% glucose, 4.5% amino acids, electrolytes, trace minerals, vitamins, and 20% fat emulsion given half-strength the first day, three-fourths strength the second day, and full strength thereafter. On postoperative day 7, surviving rats were killed. Mortality prior to day 7 was very high (68%) in the TPN group and low in the TEN (9%) and SOF (9%) groups (p < 0.005). Among survivors, the serum albumin level was lowest (p < 0.002) and serum bilirubin level (p < 0.025) and wet weight of regenerated liver (p < 0.002) highest in the TPN group. However, the livers in TPN rats appeared pale and were found to be abnormal histologically with markedly diminished glycogen and amphophylic hepatocyte cytoplasm, and their spleens were enlarged (by a factor of two). The high mortality of TPN rats was seen whether the fat emulsion was given as a bolus daily, continuously as part of the infusate, or not included as part of the TPN regimen. In the next series (n = 70), nutrient concentrations, volumes, and rates of infusion were varied. There was a high correlation between caloric (r2 = 0.831, p < 0.0006), glucose (r2 = 0.598, p < 0.02), and amino acid (r2 = 0.619, p < 0.03) intakes and mortality in the TPN group: at 140 kcal/kg/d, none died; at 178 kcal/kg/d, 50% to 62% died; and at 230 kcal/kg/d, 80% died. No TEN rat died. In conclusion, 70% hepatectomized rats fed enterally with nutrients approximating the intake of normal rats do well and survive. In sharp contrast, mortality is very high when identical nutrients are infused parenterally. By reducing the levels of nutrients given parenterally, survival improves significantly.

Ectopic expression of reg protein: a marker of colorectal mucosa at risk for neoplasia

Pancreatic regenerating gene (reg I) messenger RNA is overexpressed within the pancreas following injury and resection. Its level of expression corresponds to the level of cellular dedifferentiation. Humanreg I has been localized to chromosome 2p12, and ectopic expression of its mRNA has been found within colorectal tumors. We postulated that colorectal production ofreg I might either be a marker for the presence of cancer or, define mucosa at risk for development of neoplasia. Using a monoclonal antibody toreg I, regenerating gene protein was histochemically mapped in 56 cases of documented colorectal adenocarcinoma. In sections of colon from normal control subjects noreg I protein was noted, whereas 58.9% of the specimens from cancer patients stained positive forreg I. Although a correlation was noted betweenreg I staining and Dukes’ stage, there was no correlation with histologic grade or 5-year patient survival. In 39 of 55 cancer specimens the transition zone (interface) between the neoplasm and normal mucosa was visualized; 100% of the transition zones contained cells that stained strongly positive forreg I. We conclude thatreg I protein is ectopically expressed in colorectal mucosa at the transition zone of colorectal cancer, and occasionally within the tumor itself. Although ectopicreg I expression in colorectal epithelia is not a marker for the presence of carcinoma, it may be a sensitive marker for mucosa at risk for development of neoplasia.

DNA damage by gliotoxin from Aspergillus fumigatus. An occupational and environmental propagule: adduct detection as measured by 32P DNA radiolabelling and two-dimensional thin-layer chromatography.

Summary

Determination of plasma lactic acid concentration and specific activity using high-performance liquid chromatography

Assessment of lactate metabolism is of particular interest during exercise and in disease states such as diabetes, shock, and absorptive abnormalities of short-chain fatty acids by the colon. We describe an analytical method that introduces radio-active tracers and high-performance liquid chromatography (HPLC) to simultaneously analyze concentrations and specific activities (SAs) of plasma lactate. The HPLC conditions included separation on a reversed-phase column (octadecylsilane) and an isocratic buffer (30% acetonitrile in water). [3H]Acetate served as an internal standard. Lactate and acetate were extracted from plasma samples with diethyl ether following a pH adjustment to less than 1.0 and back-extracted into a hydrophilic phase with sodium carbonate (2 mM, pH greater than 10.0). Lactate is detected in the ultraviolet range (242 and 320 nm) by derivatization with alpha-bromoacetophenone. Control plasma samples were studied after an overnight fast for precision and analytical recovery. Calibration curves were linear in the range 0.18-6.0 mM (r = 0.92). The precision was 3% and the analytical recovery was 87%. The detection limit of the method was 36 pmol. Determination of lactate metabolism was performed in a patient with chronic congestive heart failure who was administered primed-continuous L-[U-14C]lactate (10 microCi bolus and 0.3 microCi/min continuously) during a 60-min rest period. Mean arterial lactate concentration and SA were 1.69 +/- 0.2 mM and 253.8 +/- 22 dpm/mumol, respectively. Systemic lactate turnover was 25.65 mumol/kg per min. Lactic acid systemic turnover, organ uptake and release rates can be accurately determined by isocratic HPLC.

Nonviable Staphylococcus aureus and its peptidoglycan stimulate macrophage recruitment, angiogenesis, fibroplasia, and collagen accumulation in wounded rats

We have previously shown that local application at the time of operation of Staphylococcus aureus, nonviable S. aureus, its cell wall, or S. aureus peptidoglycan accelerates wound healing. We hypothesized that this effect is due to both direct and indirect mechanisms, among which is an increase in the inflammatory response to wounding, resulting in an increase in macrophages, angiogenesis, and fibroblasts. Twenty‐seven Sprague‐Dawley male rats were anesthetized, and two 7‐cm paravertebral skin incisions were made. Four polyvinyl alcohol sponges, two on each side, containing either 100 µl of isotonic saline or 0.5 mg of nonviable S. aureus or S. aureus peptidoglycan in 100‐µl saline were implanted subcutaneously. Nonviable S. aureus or S. aureus peptidoglycan (860 µg/cm incision) in 200‐µl saline were inoculated into the incisions at closure. The rats ate a commercial rat chow and drank tap water ad libitum throughout. After days 3 and 7 postwounding, rats were euthanized, and tissues were examined for immunohistochemical features of reparative tissue using ED‐1, Factor VIII, and vimentin antibodies, markers for monocyte/macrophages, endothelial cells, and mesenchymal cells (including fibroblasts), respectively. Incisions treated with nonviable S. aureus or S. aureus peptidoglycan showed more macrophages along and deep in the wound tract 7 days postoperatively. Nonviable S. aureus or S. aureus peptidoglycan‐treated sponges were surrounded and penetrated by much larger capsules of reparative tissue than saline‐treated sponges at both 3 and 7 days. Neutrophil influx was much greater in nonviable S. aureus or S. aureus peptidoglycan‐treated sponges, especially in central regions, and there were many more ED‐1‐stained macrophages in distinct geographic locations, specifically, the more peripheral‐cortical areas. Some clustering of macrophages occurred around areas of invasion by reparative tissue into the surrounding subcutaneous fat and within the interstices of the sponges at the interface between reparative tissue and acute inflammatory cells. In contrast, saline‐treated sponge reparative tissue had significantly fewer macrophages, much thinner and flimsy reparative tissue, with proportionately fewer macrophages clustering centrally. There were many more mesenchymal cells (notably fibroblasts) and new blood vessels and much more reparative collagen in the nonviable S. aureus or S. aureus peptidoglycan‐ treated sponges. We conclude that local application of nonviable S. aureus or S. aureus peptidoglycan at wounding induces an increased number and alteration in location of macrophages, increased influx (or proliferation) of mesenchymal cells (notably fibroblasts), and increased angiogenesis and reparative collagen accumulation, as well as increasing the overall acute inflammatory response to wounding.

Contrasting effects of identical nutrients given parenterally or enterally after 70% hepatectomy: bacterial translocation.

High mortality occurs in rats with 70% hepatectomy fed intravenous (IV) total parenteral nutrition (TPN; 13.9% glucose, 4.17% amino acids, 1.46% fat, electrolytes, trace minerals, and vitamins providing 216 kcal.kg-1.d-1) but not when the identical nutrients are given at the same rate enterally (gastrostomy). We hypothesized that a difference in bacterial translocation (BT) was a contributing factor to this phenomenon. Forty-five male Sprague-Dawley rats (300-360 g) were divided into five groups and underwent the following: control (no operation), sham (intraperitoneal [IP] pentobarbital anesthesia, central venous and gastrostomy catheters, laparotomy, sham hepatectomy), standard oral feeding (SOF), TPN (IV nutrients), and total enteral nutrition (TEN; gastrostomy). The SOF, TPN, and TEN groups had IP pentobarbital anesthesia, central venous and gastrostomy catheters, and 70% hepatectomy. Postoperatively, control and SOF (both catheters plugged) rats ate a commercial rat chow and drank tap water ad libitum pre- and postoperatively. The sham, TPN, and TEN groups were given the identical infusate composition as above, but the nutrient concentrations were cut in half (110 kcal/kg) and three-quarters (165 kcal/kg) on postoperative days 1 and 2, respectively. At the end of postoperative day 2, all rats were euthanized. BT to mesenteric lymph nodes (MLNs), liver, spleen, and lungs was significantly higher in the TPN rats compared with all other groups, except that BT to the MLNs was similar in the TPN and TEN groups. Bacteremia was found only in the TPN rats. BT in TPN rats with 70% hepatectomy was significantly greater 48 h after operation than in those fed the identical nutrients enterally at the same rate; this correlates with the previously reported significantly greater mortality in rats with 70% hepatectomy receiving TPN.

Cisplatin DNA Adduct Detection and Depurination Measured by 32P DNA Radiolabeling and Two-Dimensional Thin-Layer Chromatography: A Time and Concentration Study

Abstract Platinum-based chemotherapies cause the formation of DNA adducts and have profound effects on DNA. This study measured cis-diamminedichloroplatinum II (cisplatin) DNA adducts by 32P-radiolabeling DNA, enzymatically digesting radiolabeled DNA, separating the formed adducts on two-dimensional thin-layer chromatography, and quantitating the adducts with autoradiography and densitometry. HeLa DNA was incubated with cisplatin at varying concentrations (6.25–325 nM) and times (0 min to 72 hr). Cisplatin rapidly depurinated dGMP and dAMP (90%, 15–min incubation with 325 nM cisplatin). Partial depurination of dGMP (15%) and dAMP (25%) occurred with lower cisplatin concentrations at equal incubation times. a minimum of four new adducts, with relatively rapid migratory patterns, were detected at high cisplatin concentrations with short incubation times. These results indicate that the depurination of DNA correlates with DNA adduct formation and that the quantification of these adducts may be applicable to monitoring tumor and host cell response to cisplatin chemotherapy.

Staphylococcus aureus peptidoglycan ameliorates cyclophosphamide-induced impairment of wound healing.

Cyclophosphamide given systemically to rats leads to impaired wound healing, characterized by decreases in the inflammatory reaction, fibroplasia, neovascularization, reparative collagen accumulation, and wound breaking strength. In contrast, the local application of Staphylococcus aureus peptidoglycan at the time of wounding increases all of these processes in normal rats. Accordingly, we hypothesized that inoculation of S. aureus peptidoglycan into wounds of cyclophosphamide‐treated rats would ameliorate the otherwise impaired healing. Dorsal bilateral skin incisions and subcutaneous implantation of polyvinyl alcohol sponges (two on each side) were performed on male Sprague‐Dawley rats receiving either saline or cyclophosphamide (24 mg/kg) intraperitoneally at the time of operation, on postoperative days 1, 2, 3, 4 (for rats killed on postoperative day 7), and also on day 8 (for rats killed on postoperative day 14). The incisions on one side were inoculated at the time of closure with 0.2 ml of saline solution, and the incisions on the other side with 6 mg S. aureus peptidoglycan in 0.2 ml saline solution (860 µg/cm incision). The sponges were instilled with 0.1 ml saline solution on the saline solution‐instilled incision side or with S. aureus peptidoglycan 0.5 mg/sponge) in 0.1 ml saline solution on the other side. In control rats receiving saline solution intraperitoneally, incisions treated with S. aureus peptidoglycan were significantly stronger than saline solution‐treated incisions by a factor of 1.8 at 1 week (p < 0.001); at 2 weeks the increase was small and not significant. Cardiac blood leukocytes and platelets fell markedly (90%) in cyclophosphamide‐ treated rats, and there was a decrease in wound breaking strength of their saline‐treated incisions at both 7 and 14 days compared with saline solution‐treated incisions of control rats. S. aureus peptidoglycan treatment of the wounds completely prevented this effect at 7 days, and partially at 14 days. Polyvinyl alcohol sponge reparative tissue hydroxyproline, 7 days after surgery, was decreased in cyclophosphamide‐treated rats; this was completely prevented by S. aureus peptidoglycan treatment of the sponges. Histologically, the inflammatory response to the wounding, influx of macrophages and fibroblasts, angiogenesis, and collagen accumulation were all reduced at day 7 and 14 after surgery in the sponge reparative tissue of cyclophosphamide‐ treated rats; this was prevented by S. aureus peptidoglycan treatment of the sponges. In conclusion, a single local application of S. aureus peptidoglycan ameliorates cyclophosphamide‐impaired wound healing.

Single local instillation of nonviable Staphylococcus aureus or its peptidoglycan ameliorates glucocorticoid‐induced impaired wound healing

An excess in glucocorticoid steroids, either from endogenous or exogenous sources, has been shown to inhibit wound repair. Key to this impairment is a diminution of the inflammatory response to wounding, fibroplasia, capillary formation, reparative tissue collagen accumulation, and wound breaking strength. Because a single local application at operation of nonviable Staphylococcus aureus or its peptidoglycan increases all of these processes in normal rats, we hypothesized that nonviable S. aureus and S. aureus peptidoglycan would each ameliorate glucocorticoid‐induced impaired healing. Sprague‐Dawley male rats aseptically received two 7 cm paravertebral skin incisions and underwent subcutaneous implantation of polyvinyl alcohol sponges. Two glucocorticoids were used: hydrocortisone, 8 mg intramuscularly, daily beginning 1 day before operation and continuing during the postoperative period; or a single dose of a long‐acting preparation of methylprednisolone, 6 or 8 mg intramuscularly, on the day before operation. Controls received intramuscular injections of saline solution at the same respective times. At the time of the operation, one incision and the polyvinyl alcohol sponges on one side of the animal were instilled with saline solution while the incision and sponges on the opposite side were instilled with nonviable S. aureus (hydrocortisone study) or S. aureus peptidoglycan (two methylprednisolone studies). The data showed that, at postoperative day 7, the single local application at wounding of nonviable S. aureus or S. aureus peptidoglycan increased wound breaking strength in the control rats by factors of 1.6 in the hydrocortisone experiment and 1.4 and 1.6 in the methylprednisolone studies. These treatments prevented (in hydrocortisone‐treated rats) or mitigated (in methylprednisolone‐treated rats) the glucocorticoid‐induced decrease in wound breaking strength. In addition, these treatments prevented the glucocorticoid‐induced decreases in the inflammatory (largely mononuclear cells) response to wounding and in the accumulation within the polyvinyl alcohol sponge of reparative tissue fibroblasts, capillaries, and collagen.