Purified human breast milk MUC1 and MUC4 inhibit human immunodeficiency virus.

BACKGROUND: The HIV-AIDS pandemic is prevalent in sub-Saharan Africa. Breastfeeding is a risk factor, with transmission from mother to child being as high as 40%. OBJECTIVES: To determine the antiviral activity of crude breast milk and its purified mucins MUC1 and MUC4 against HIV-1 in patients who were HIV positive compared to those who were not. METHODS: Twenty-one human milk samples were taken from both groups. Breast milk mucins were purified by density-gradient ultracentrifugation in caesium chloride and analyzed by SDS-PAGE, Western blotting and amino acid content. The inhibition of the virus by crude milk and purified mucin was assayed by an in vitro HIV-1 p24 assay. RESULTS: SDS-PAGE for purified mucin showed several high-molecular-weight bands for the HIV-negative group and prominently stained single bands on the stacking gel with faintly periodic acid Schiff-positive glycoprotein bands observed in some cases in the running gel for the HIV-positive mucins. Western blot analysis identified the mucins in both groups to be MUC1 and MUC4. Both mucins showed more intensity on Western blotting for the HIV-positive group. There was no difference in the content of serine, threonine and proline of purified mucins for both groups. HIV-1 was not inhibited by crude breast milk from normal (13/14 samples) and infected individuals (19/19 samples). Fifteen of 20 and 16/18 samples of purified mucin from the uninfected and HIV-positive groups, respectively, inhibited the virus. CONCLUSIONS: Crude breast milk does not inhibit HIV-1, whilst purified mucins do in an in vitro assay.

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 a-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.

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.

Vaccinia virus complement control protein (VCP) improves kidney structure and function following ischemia/reperfusion injury in rats.

Renal transplantation is often confronted with ischemia reperfusion (I/R) injury that accounts for a delayed recovery of the graft. This surgically and biologically induced injury often results in activation of the complement system. The vaccinia virus complement control protein (VCP) down-regulates both the classical and alternative complement pathways by preventing the formation of C3b, a component where both pathways converge. The aim of the study was to investigate the effect of VCP on renal I/R injury. Long Evans rats were subjected to laparotomy, mobilization of the right kidney in unilateral ischemia, and both kidneys in bilateral ischemia. The renal arteries were clamped for 60 min followed by 24 h reperfusion time. The animals were randomly allocated to receive recombinant VCP (rVCP), natural VCP, and humanized recombinant VCP (hrVCP) combination, vehicle (PBS), or sham group. Blood samples were collected for biochemical studies, and the kidneys were removed for histopathologic and immunohistochemical studies. The biochemical studies in the bilateral ischemia showed that the PBS group displayed 1.5-fold and 5-fold increases in the urea and creatinine concentrations, respectively, compared with the VCP/hrVCP groups. In both models, the histopathologic study revealed focal necrosis of the tubular epithelial cells in the rVCP or VCP/hrVCP treated animals compared with the diffuse and markedly elevated field scores in the PBS controls. The immunohistochemical study showed significant C3 deposition in the renal tubules of the PBS controls compared with the rVCP or VCP/hrVCP groups, suggesting that rVCP, VCP/hrVCP reduced I/R injury by inhibiting the biosynthesis of C3.

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

BACKGROUND: The 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. METHODS: Pregnancy 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). RESULTS: The 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. CONCLUSION: Although 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.

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.

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 patient's 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.

The characterisation of mucin in a mature ovarian teratoma occurring in an eight year old patient.

INTRODUCTION: The presence of MUC5AC (M1 antigen) and MUC6 have previously been found in ovarian mucinous cyst. We characterized the mucins in the crude mucus and tissue of a mature ovarian teratoma in an 8 year old girl. MATERIALS AND METHODS: Mucins were purified from crude mucus by density gradient ultra-centrifugation in CsCl and analysed by gel-filtration and SDS-PAGE analysis. Mucin identification and expression was by western blotting and immunohistochemistry. RESULTS: Histology showed a tumour with solid and cystic areas, with the cysts lined by colonic and respiratory mucosae. Equal volumes of 'sol' and 'gel' phases of approximately 10.0 ml of crude mucus were obtained. Gel filtration and SDS-PAGE analyses suggested that the mucin was mainly of the large polymeric type which dissociated upon reduction of disulphide bonds with DTT. The colonic and respiratory epithelia predominantly expressed acidic mucin of the sialated and sulphated types respectively. MUC1 and MUC1c were expressed exclusively in respiratory epithelium, MUC2 and some MUC6 (focal) in the colonic tissue and MUC5AC in both tissues. Western blotting confirmed the presence of MUC2, MUC5AC and MUC5B in the secreted gel. Serine, threonine and proline made up the bulk of the amino acids in the sample. DISCUSSION: Ovarian teratoma produced a highly viscous mucus secretion in which the mucin was largely polymeric and of the MUC2, MUC5AC and MUC5B type. The respiratory component of the teratoma expressed MUC1 and MUC1c and the colonic components of the teratoma expressed MUC2 and some MUC6. MUC5AC was expressed in both components.

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.

The relationship of a Helicobacter heilmannii infection to the mucosal changes in abattoir and laboratory pig stomach.

PURPOSE: A pig ulcer model in which ulceration is reproducibly induced in the pars oesophagea (a tongue of the oesophageal squamous epithelium that extends into the pig stomach) by bile duct ligation (BDL) was used in this study to determine whether Helicobacter heilmannii (Hh) is a predisposing factor in the ulceration of this region. The infection with Hh and its relationship to ulceration and mucus integrity was examined. METHODS: We microscopically investigated the occurrence of spontaneous pars oesophageal ulceration in 33 pigs from a local abattoir and 5 pigs nurtured in pens in our surgical laboratory (JSM). Further groups of 5 and 6 JSM pigs underwent a sham operation and a BDL, respectively. Giemsa staining was used to detect Hh and purified mucin was characterized by gel filtration. RESULTS: Ten of 33 and 2 of 5 of the stomachs of abattoir and JSM pigs, respectively, were positive for Hh by Giemsa stain. Three of the 33 abattoir pigs showed ulceration in the pars oesophagea and none of these was Hh-positive. All six of the bile duct-ligated pigs showed ulceration in the pars but only 2 of these were Giemsa-positive. Only 8 of 33 of the abattoir pigs had > or =50% large polymeric mucin that was eluted in the void/excluded volume of a Sepharose 2B column. CONCLUSIONS: There was no consistent correlation between an infection of the pig stomachs by Hh, an ulceration of the pars oesophagea, and mucin degradation. There was a significant difference between the percentage of polymeric mucin from the abattoir pigs and that of the JSM group (P < 0.003), the JSM group vs sham-operated pigs (P < 0.011), and JSM vs BDL pigs (P < 0.0005), but there appeared to be no association between the infectivity with Hh and mucin degradation.

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.