Marilyn Tyler

Inhibition of Human Immunodeficiency Virus Type 1 Activity by Purified Human Breast Milk Mucin (MUC1) in an Inhibition Assay

It has been reported that breast-feeding is responsible for approximately 40% of the HIV transmissions from HIV-positive mothers to children. Human breast milk, however, is known to contain numerous biologically active components which protect breast-fed infants against bacteria, viruses, and toxins. The purpose of this study was to purify and characterize breast milk mucin and to determine its anti-HIV-1 activity in an HIV inhibition assay. Sepharose CL-4B column chromatography and caesium chloride isopycnic density gradient purification were used to isolate and purify the mucin. Following Western blotting and amino acid analysis, an HIV-1 inhibition assay was carried out to determine the anti-HIV-1 activity of crude breast milk and purified milk mucin (MUC1) by incubating them with HIV-1 prior to infection of the human T lymphoblastoid cell line (CEM SS cells). SDS-PAGE analysis of the mucin, together with its amino acid composition and Western blotting, suggested that this purified mucin from human breast milk was MUC1. The HIV inhibition assay revealed that while the purified milk mucin (MUC1) inhibited the HIV-1 activity by approximately 97%, there was no inhibition of the HIV-1 activity by crude breast milk. Although the reason for this is not clear, it is likely that because the MUC1 in crude milk is enclosed by fat globules, there may not be any physical contact between the mucin and the virus in the crude breast milk. Thus, there is a need to free the mucin from the fat globules for it to be effective against the virus.

Antiviral Activity of Purified Human Breast Milk Mucin

Human breast milk is known to contain numerous biologically active components which protect breast fed infants against microbes, viruses, and toxins. The purpose of this study was to purify and characterize the breast milk mucin and determine its anti-poxvirus activity. In this study human milk mucin, free of contaminant protein and of sufficient quantity for further analysis, was isolated and purified by Sepharose CL-4B gel filtration and cesiumchloride density-gradient centrifugation. Based on the criteria of size and appearance of the bands and their electrophoretic mobility on sodium dodecyl sulfate polyacrylamide-gel electrophoresis, Western blotting together with the amino acid analysis, it is very likely that the human breast milk mucin is MUC1. It was shown that this breast milk mucin inhibits poxvirus activity by 100% using an inhibition assay with a viral concentration of 2.4 million plaque-forming units/ml. As the milk mucin seems to aggregate poxviruses prior to their entry into host cells, it is possible that this mucin may also inhibit other enveloped viruses such as HIV from entry into host cells.

Energy charge as an indication of liver viability : a comparison of changes in livers that remained intact with those subjected to autografting

As efforts to assess the viability of liver grafts continue, the recent description of noninvasive measurement by fluorimetry or magnetic resonance spectrometry of adenine nucleotides has brought energy charge into focus again as an index of viability. Many previous studies have been conducted in human donor livers that have clinical relevance but which cannot be standardized, or in rats in which the hepatic artery is not anastomosed. In the present study, pig livers were definitively rendered ischemic for 1 or 2 hr. In one group the livers were then revascularized (intact) while in the other, the livers were removed during the final 20-30 min of the ischemic period and were subjected to autograft. There was a marked difference in survival between the intact and the autograft groups. One hour of ischemia in the intact group was associated with survival comparable to that of autograft controls (8-100 days); 2 hr of ischemia caused shortened survival, ranging from 2 to 18 days. In the recipients of autografts, survival after 1 hr of ischemia ranged from 3 to 16 days; after 2 hr of ischemia no autograft recipient survived overnight. The energy charge returned to the preoperative level after 2 hr of ischemia in both intact and autograft groups. The concentrations remained depressed after 2 hr of ischemia in autografted animals, thus being associated with survival. However, the patterns of total adenine nucleotide and adenosine triphosphate were not always similar to those of energy charge. The concentrations of aspartate aminotransferase were similarly elevated in all ischemic groups irrespective of duration or subsequent survival. There was, however, a close association between euglobulin lysis times (ELT) and survival. In the autograft recipients of livers subjected to 2 hr of ischemia that did not survive overnight the ELT remained significantly shortened. It is concluded that adenine nucleotide metabolism is important as an index of viability, but that concentrations of total and individual adenine nucleotides and the energy change all need to be computed. There does, however, appear to be an absolute relationship between survival and euglobulin lysis time that would be clinically useful in patients undergoing liver transplantation or hepatic vascular exclusion.

The expression of MUC mucin in cholangiocarcinoma

Cholangiocarcinoma (CC) is a highly malignant epithelial cancer of the biliary tract, the cellular and molecular pathogenesis of which remains unclear. Malignant transformation of glandular epithelial cells is associated with the altered expression of mucin. We investigated the type of mucins expressed in CC. Twenty-six patients with histologically confirmed CC were included in this study. The expression of mucin was studied by immunohistochemistry using antibodies to MUC1, MUC1 core, MUC2, MUC3, MUC4, MUC5AC, and MUC6. There was extensive (>50%) expression of mucin, mainly MUC1 in 11/25 and MUC5AC in 12/26 cases. In the case of MUC3, 6/26 cases expressed mucin extensively, whilst only 1/26 had MUC2, MUC4, and MUC6 expression. Well-differentiated tumors significantly expressed MUC3 extensively compared to poor or moderately differentiated tumors (p=0.003). Fifteen of 25 cases had metastatic disease. MUC1 was extensively expressed in 9/15 cases with metastatic disease. In contrast, MUC1 expression was present in 2/10 cases where metastases were absent. Hilar lesions were less likely to express MUC1, but this was not statistically significant. Fifteen of 25 cases had metastatic disease. Extensive MUC3 expression was significantly associated with well-differentiated tumors, whilst there was an approaching significance between the extensive expression of MUC1 and metastasis in cholangiocarcinoma.

The inhibition of the Human Immunodeficiency Virus type 1 activity by crude and purified human pregnancy plug mucus and mucins in an inhibition assay

BackgroundThe female reproductive tract is amongst the main routes for Human Immunodeficiency Virus (HIV) transmission. Cervical mucus however is known to protect the female reproductive tract from bacterial invasion and fluid loss and regulates and facilitates sperm transport to the upper reproductive tract. The purpose of this study was to purify and characterize pregnancy plug mucins and determine their anti-HIV-1 activity in an HIV inhibition assay.MethodsPregnancy plug mucins were purified by caesium chloride density-gradient ultra-centrifugation and characterized by Western blotting analysis. The anti-HIV-1 activities of the crude pregnancy plug mucus and purified pregnancy plug mucins was determined by incubating them with HIV-1 prior to infection of the human T lymphoblastoid cell line (CEM SS cells).ResultsThe pregnancy plug mucus had MUC1, MUC2, MUC5AC and MUC5B. The HIV inhibition assay revealed that while the purified pregnancy plug mucins inhibit HIV-1 activity by approximately 97.5%, the crude pregnancy plug mucus failed to inhibit HIV-1 activity.ConclusionAlthough it is not clear why the crude sample did not inhibit HIV-1 activity, it may be that the amount of mucins in the crude pregnancy plug mucus (which contains water, mucins, lipids, nucleic acids, lactoferrin, lysozyme, immunoglobulins and ions), is insufficient to cause viral inhibition or aggregation.

MUC2, MUC5AC and MUC5B in the mucus of a patient with pseudomyxoma peritonei: Biochemical and immunohistochemical study

A 58‐year‐old man with a 1 year history of progressive abdominal distension underwent a laparotomy for pseudomyxoma peritonei. The mucin was identified and characterized in the present study. Approximately 6 L of crude mucus in the sol (highly viscous) and gel (semisolid) phases was obtained from the patients peritoneal cavity. The sol material was briefly homogenized followed by slow stirring at dilutions of up to 1:10 with 6 mol/L guanidinium chloride and proteolytic inhibitors for periods of up to 48 h. Preparative and analytical gel filtration on Sepharose 2B showed some PAS‐positive material eluting in the void volume accompanied by equal or larger amounts of protein in the void and included volumes of the columns. Sodium dodecylsulfate–polyacrylamide gel electrophoresis of purified mucin on a 4–20% gradient gel showed PAS‐positive material on the top of the running gel and a distinct smaller‐sized species of mucin of higher electrophoretic mobility with background material in between the large and small mucin. Western blot (confirmed by immunohistochemical analysis) after agarose gel electrophoresis showed the presence of MUC2, MUC5AC and MUC5B in the mucus. There was no MUC1, MUC1core or MUC6 in the tissue. Histopathological examination confirmed a mucinous appendicular adenocarcinoma. Histology showed the mucin to be predominantly of the sulfated and non‐sulfated acidic type. Serine, threonine and proline comprised 21.6% of the total amino acid composition of the sample. The viscous nature of the material is due to the presence of three gel‐forming mucins and possibly to its high content of protein.

Effect of total hepatectomy on coagulation and glucose homeostasis in the pig.

It has been suggested recently that patients with fulminant liver failure should be prepared for transplantation by early hepatectomy, yet the acute effects of removal of the liver upon the coagulation profile and certain hormones are not known. This study was conducted on totally hepatectomized pigs that survived up to 27 hr. Measurements were made of serum insulin, plasma glucagon (IRG and GLI), glucose, catecholamines, and the coagulation profile. The increase in serum insulin was directly related to levels of plasma glucosethere was a 100-fold increase in animals with plasma glucose levels>400 mg/100ml and none when blood glucose was <100 mg/100 ml. Plasma glucagon showed a sharp transient increase within 1 hr of hepatectomy and a slow rise thereafter with levels apparently unrelated to serum insulin or plasma glucose. There was a transient increase in plasma adrenaline but a sharp continous increase in plasma norepinephrine. No changes of note occurred in the coagulation profile-even levels of fibrinogen only declined by 20% in 27 hr. The study has shown that early total hepatectomy is safe as far as changes in coagulation are concerned but changes in serum insulin and especially plasma norepinephrine may be of more significance.

REPERFUSION INJURY ASSOCIATED WITH PORTAL VENOUS AND HEPATIC ARTERIAL PERFUSION IN LIVER TRANSPLANTATION

Background. Although reperfusion injury in organ transplantation is presently well established, its exact role in liver transplantation still has to be defined. The aim of this part of the study was to document the reperfusion injury associated with porcine liver transplantation and to evaluate the different components of the reperfusion injury associated with arterial and portal reperfusion. Methods. Large white X Landrace pigs were randomized into two groups: group 1, initial portal reperfusion, and group 2, initial arterial reperfusion. Several indicators of reperfusion injury, endothelial cell function, and hepatocellular damage were assessed. Early histopathological findings in biopsy specimens can predict poor graft outcome, therefore histological findings of the liver biopsies after liver transplantation were also studied. Results. Malondialdehyde concentrations were lower and vitamin A concentrations were higher in the animals subjected to initial portal reperfusion. Serum amino aspartate transferase and serum hyaluronic acid concentrations were higher in the animals subjected to initial portal reperfusion. Histological results showed that hepatocyte vacuolization, neutrophil infiltration, single hepatocyte necrosis, and group cell necrosis of the hepatocytes were all significantly reduced in group 2 compared to group 1. Conclusion. Results of this study indicate that the major part of reperfusion injury is constituted during the portal venous reperfusion and that this injury can be, at least partially, attenuated by initial arterial reperfusion.

A 40-50kDa Glycoprotein Associated with Mucus is Identified as α-1-Acid Glycoprotein in Carcinoma of the Stomach

Background and Aim: Secreted gastric mucins are large O-glycosylated proteins of crude mucus gels which are aberrantly expressed in malignancy. An albumin associated 55-65kDa glycoprotein was previously shown in mucus gels in gastric cancer. The aim of this study was to investigate its expression and identification in human gastric tissue. Methods: Mucins were purified from crude mucus scrapings of 16 partial and 11 total resections and a rabbit polyclonal antibody was raised to the 55-65kDa glycoprotein. The location and expression of the glycoprotein was examined in normal gastric mucosa (n=20), intestinal metaplasia (n=18) and gastric cancer (n=27) tissue by immunohistochemistry. Mucins were analyzed by isoelectric focusing (IEF) on 2-D polyacrylamide gels. Identification of the 40-50kDa glycoprotein was by MALDI-TOF MS technique. Plasma levels were examined by Western blotting. Results: Extensive SDS-PAGE analysis gave a PAS positive glycoprotein in the 40-50kDa range, in patients with gastric cancer but not normals. It was expressed in parietal and columnar cells of normal gastric tissue and intestinal metaplasia respectively, and in 22 of 27 gastric cancer specimens. In 2-D PAGE stained with Coomassie Blue there were 3 spots positively identified as alpha-1-acid glycoprotein (AGP) by MALDI-TOF MS technique. PAS staining revealed a single bright spot in the same position but could not be identified. Preliminary measurements showed slightly higher levels of AGP in plasma of patients with gastric carcinoma. Conclusion: AGP levels are increased in gastric tissue and in the plasma of those with carcinoma of the stomach.

Immunohistochemical and Biochemical Characterization of Mucin in Pseudomyxoma Peritonei: A Case Study

We previously reported the presence of MUC2, MUC5AC and, for the first time, MUC5B in a 58-year-old male with pseudomyxoma peritonei (PMP). This is a report on the biochemical and immunohistochemical characterization of mucin in a 50-year-old female with the same rare illness. A right oophorectomy and appendicectomy and a resection of the involved omentum were performed. Approximately a litre of crude material in the sol and gel phases was obtained from the patient during laparotomy. This was briefly homogenized in 6 M guanidinium hydrochloride and proteolytic inhibitors and purified by density gradient centrifugation in caesium chloride. At laparotomy it was noted that the patient had appendiceal and ovarian masses as well as extensive mucinous deposits in the omentum and peritoneum. A mucinous adenocarcinoma of the appendix and ovary was confirmed on histology. The cells expressed both sulphated and non-sulphated acidic mucins. The presence of MUC2, MUC5AC, MUC5B and α-1-acid glycoprotein was shown by Western blotting and MUC4 by immunohistochemical staining. MUC1 and MUC6 were not detectable in the tissue. The study confirms that MUC2, MUC5AC and MUC5B are produced in the mucus of patients with PMP. The expression of MUC4 in this disease has not been previously reported.