Treatment and outcomes of two pigs treated for hemoabdomen secondary to splenic disease.

OBJECTIVE: To report the management and outcomes of two pigs undergoing emergency surgery for hemoabdomen secondary to splenic disease. STUDY DESIGN: Case report. ANIMAL: Two adult pigs with hemoabdomen and suspected splenic pathology. METHODS: Pigs were admitted for several clinical signs including lethargy, inappetence, vomiting, abdominal distention, hypothermia, and tachycardia. Abdominal ultrasound and abdominocentesis led to a diagnosis of hemoabdomen secondary to splenic disease. RESULTS: The spleen was confirmed as the source of hemorrhage during midline exploratory celiotomy in both pigs. Splenic rupture resulted from splenic vein thrombosis in one pig and splenic torsion in the other. Complications included intraoperative hemorrhage and intraabdominal adhesion formation. Four years following splenectomy, one pig was euthanized because of widespread small intestinal adhesions causing luminal obstruction, while the other pig was euthanized following a diagnosis of chronic myeloid leukemia. CONCLUSIONS: A ventral midline celiotomy provided adequate exposure for splenectomy. The procedure allowed resolution of signs in both pigs. Splenic pathology, such as vessel thrombosis or torsion, may result in splenic rupture and should be considered as a differential in pigs with hemoabdomen.

Rationale and methods for a cross-sectional study of mental health and wellbeing following river flooding in rural Australia, using a community-academic partnership approach.

BACKGROUND: Climate change is associated with greater frequency, duration, intensity and unpredictability of certain weather-related events, including floods. Floods harm mental health. There is limited understanding of the mental health and well-being effects from river flooding, particularly over the longer term and in rural contexts. This paper describes the rationale, aims, objectives, study design and socio-demographic characteristics of the sample for a study measuring associations between flood experience and mental health and wellbeing of residents (particularly those most likely to be negatively impacted and hard to reach) in rural NSW Australia 6 months following a devastating flood in 2017. To our knowledge, the study is the first of its kind within Australia in a rural community and is an important initiative given the likelihood of an increasing frequency of severe flooding in Australia given climate change. METHODS: A conceptual framework (The Flood Impact Framework) drawing on social ecological approaches was developed by the research team. It was based on the literature and feedback from the community. The Framework describes putative relationships between flood exposure and mental health and wellbeing outcomes. Within a community-academic partnership approach, a cross-sectional survey was then undertaken to quantify and further explore these relationships. RESULTS: The cross-sectional survey was conducted online (including on mobile phone) and on paper between September and November 2017 and recruited 2530 respondents. Of those, 2180 provided complete demographic data, among whom 69% were women, 91% were aged 25-74, 4% identified as Aboriginal and/or Torres Strait Islander, 9% were farmers and 33% were business owners. CONCLUSIONS: The study recruited a wide range of respondents and the partnership facilitated the community's engagement with the design and implementation of the study. The study will provide a basis for a follow-up study, that will aim to improve the understanding of mental health and wellbeing effects over the longer term. It will provide an important and original contribution to understanding river flooding and mental health in rural Australia, a topic that will grow in importance in the context of human-induced climate change, and identify critical opportunities to strengthen services, emergency planning and resilience to future flooding.

Transnasal, Endoscopically Guided Skull-Based Surgery by Pharyngotomy for Mass Removal from the Sphenopalatine Sinus in a Horse.

OBJECTIVE: To report a transnasal, endoscopically guided ventral surgical approach for accessing the cranial and caudal segments of the sphenopalatine sinus for mass removal in a horse. STUDY DESIGN: Case report. ANIMAL: Adult horse with acute onset blindness referable to a soft tissue mass within the sphenopalatine sinus. CLINICAL REPORT: A 7-year-old Warmblood gelding presented with a history of running into a fence and falling. No neurologic signs were identified at initial examination but acute blindness was noted 3 weeks later. On computed tomography (CT) the sphenopalatine sinus was filled with a large homogeneous mass with poor contrast enhancement that extended dorsally with thinning to the dorsal cortex of the sphenoid bone, just rostral to the entrance of the optic canals into the cranial cavity. Surgical access to the sphenopalatine sinus was achieved using a transnasal, endoscopically guided ventral pharyngotomy approach and the mass lesion was removed. A presumptive diagnosis of chondroma was made based on histopathology. The horse recovered well from surgery, and although it has not regained vision as of 6.5 years postoperatively, the disease has not progressed. CONCLUSION: Transnasal, endoscopically-guided ventral surgical access to the sphenopalatine sinus is possible in horses and may improve access in horses with disease extending caudally beyond the palatine portion of the sinus. Use of smaller diameter or specialized instruments, such as various endoscopic bone cutting instruments, and CT image guidance may improve sinus access by this route.

Laparoscopic-guided compared to skilled instructor support for student rectal examination training using live horses in the veterinary curriculum.

OBJECTIVES: To evaluate the veterinary student learning outcome of 2 methods of equine rectal examination training. STUDY DESIGN: Randomized prospective study. SAMPLE POPULATION: Veterinary students (3rd and 4th year; n = 40) and practicing equine veterinarians (n = 10). METHODS: Year 1: Group 1 (n = 11) and Group 2 students (n = 10) received skilled instructor (SI) and laparoscopic-guidance (LG), respectively, during rectal exam instruction. All students were tested on rectal identification of 4 abdominal organs. Year 2: One group of students (n = 19) was trained and subsequently tested using each technique, first SI, followed by LG. Subjective evaluation of laparoscopy as a teaching tool was achieved with veterinary students and equine practitioners. RESULTS: A significantly greater percentage of students having LG compared to SI were able to correctly identify the left kidney (Year 1) and the spleen, cecum, and right ovary (Year 2). A significantly greater proportion of LG trained students in years 1 and 2 (100% and 95%, respectively) were also able to identify 75% of organs compared with SI (27% and 21%, respectively). Both students and veterinarians uniformly provided favorable feedback for LG in teaching rectal palpation skills. CONCLUSION: The LG method of equine rectal examination instruction resulted in improved learning for identification of several key abdominal organs compared with SI.

Interpreting abdominal fluid in colic horses: Understanding and applying peritoneal fluid evidence.

BACKGROUND: Interpreting changes in peritoneal fluid helps clinicians manage colic and other diseases in horses. During abdominal problems in the horse, abdominal fluid characteristics such as color, turbidity, total nucleated and red blood cell counts, cytology, total protein, and l-lactate change in predictable ways, helping the clinician characterize the disease. DESCRIPTION: Normal abdominal fluid in horses is odorless, clear to light yellow in color, and transparent. Peritoneal fluid becomes more turbid with increasing levels of protein, number of WBCs or RBCs, or with gross contamination following intestinal rupture. The color of abdominal fluid will also change with the type and quantity of cells or other elements present. The transformation of peritoneal fluid color from golden to orange to red represents increasing levels of RBCs, common with strangulating intestinal lesions. Serosanguinous defines fluid that is both turbid and orange to bloody because of increased total protein, WBCs, and RBCs, and is considered classic for diseases characterized by intestinal ischemia. Peritoneal fluid may also be red or blood-colored because of a hemoperitoneum, or secondary to blood contamination during sample collection. l-Lactate measurement in the abdominal fluid has proven invaluable for the identification of strangulating intestinal injury. Cytology acts as an important supplement to cell counts in peritoneal fluid, and the normal ratio of non-degenerate neutrophils:mononuclear cells of 2:1 changes during various gastrointestinal diseases. Culture of peritoneal fluid samples should be performed when septic peritonitis is suspected. SUMMARY: Abdominal fluid is a sensitive indicator of intestinal injury and a useful tool to direct treatment. Peritoneal fluid evaluation includes gross visual and olfactory examination, nucleated cell count, total protein, RBC count, lactate levels, cytology, and culture. The changes noted in such variables are related to the type and duration of the abdominal problem. KEY POINTS:  Abdominal fluid interpretation has become central to the triage and management of challenging equine colic patients.  The transformation of peritoneal fluid color from golden to orange to red represents increasing levels of RBCs, common with strangulating intestinal lesions.  Contamination with RBCs at various concentrations may be secondary to vascular (eg, abdominal wall or mesenteric vessels) or splenic trauma during abdominal fluid collection; however, this must be distinguished from orange to red fluid associated with intestinal strangulating obstruction or hemoabdomen  Peritoneal fluid analysis reveals abdominal pathology by recognizing specific changes that occur with disease processes affecting the tissues and organs within this cavity.  Abdominal fluid examination should be used as a tool to direct treatment rather than the definitive test for diagnosis of the acute abdomen  Septic peritonitis in horses most commonly originates secondary to intestinal compromise or accidents (vascular damage, perforation, or surgical manipulation), leading to bacterial translocation into the abdomen.

Calculating and selecting fluid therapy and blood product replacements for horses with acute hemorrhage.

BACKGROUND: Blood products, crystalloids, and colloid fluids are used in the medical treatment of severe hemorrhage in horses with a goal of providing sufficient blood flow and oxygen delivery to vital organs. The fluid treatments for hemorrhage will vary depending upon severity and duration and whether hemorrhage is controlled or uncontrolled. DESCRIPTION: With acute and severe controlled hemorrhage, treatment is focused on rapidly increasing perfusion pressure and blood flow to vital organs. This can most easily be accomplished in field cases by the administration of hypertonic saline. If isotonic crystalloids are used for resuscitation, the volume administered should be at least as great as the estimated blood loss. Following crystalloid resuscitation, clinical signs, HCT, and laboratory evidence of tissue hypoxia may help determine the need for a whole blood transfusion. In uncontrolled hemorrhage, crystalloid resuscitation is often more conservative and is referred to as "permissive hypotension." The goal of "permissive hypotension" would be to provide enough perfusion pressure to vital organs such that function is maintained while keeping blood pressure below the normal range in the hope that clot formation will not be disrupted. Whole blood and fresh frozen plasma in addition to aminocaproic acid are indicated in most horses with severe uncontrolled hemorrhage. SUMMARY: Blood transfusion is a life-saving treatment for severe hemorrhage in horses. No precise HCT serves as a transfusion trigger; however, an HCT < 15%, lack of appropriate clinical response, or significant improvement in plasma lactate following crystalloid resuscitation and loss of 25% or more of blood volume is suggestive of the need for whole blood transfusion. Mathematical formulas may be used to estimate the amount of blood required for transfusion following severe but controlled hemorrhage, but these are not very accurate and, in practice, transfusion volume should be approximately 40% of estimated blood loss. KEY POINTS: Modest hemorrhage, <15% of blood volume (<12 mL/kg), can be fully compensated by physiological mechanisms and generally does not require fluid or blood product therapy. More severe hemorrhage, >25% of blood volume (> 20 mL/kg), often requires crystalloid or blood product replacement, while acute loss of greater than 30% (>24 mL/kg) of blood volume may result in hemorrhagic shock requiring resuscitation treatments Uncontrolled hemorrhage is a common occurrence in equine practice, and is most commonly associated with abdominal bleeding (eg, uterine artery rupture in mares). If the hemorrhage can be controlled such as by ligation of a bleeding vessel, then initial efforts to resuscitate the horse should focus on increasing perfusion pressure and blood flow to organs as quickly as possible with crystalloids or colloids while assessing need for whole blood transfusion. While fluid therapy is being administered every effort to physically control hemorrhage should be made using ligatures, application of compression, surgical methods, and local hemostatic agents like collagen-, gelatin-, and cellulose-based products, fibrin, yunnan baiyao (YB), and synthetic glues Although some synthetic colloids have been shown to be associated with acute kidney injury in people receiving resuscitation therapy,20 this undesirable effect in horses has not been reported.

The pathophysiology of uncontrolled hemorrhage in horses.

BACKGROUND: Hemorrhagic shock in horses may be classified in several ways. Hemorrhage may be considered internal versus external, controlled or uncontrolled, or described based on the severity of hypovolemic shock the patient is experiencing. Regardless of the cause, as the severity of hemorrhage worsens, homeostatic responses are stimulated to ameliorate the systemic and local effects of an oxygen debt. In mild to moderate cases of hemorrhage (<15% blood volume loss), physiological adaptations in the patient may not be clinically apparent. As hemorrhage worsens, often in the uncontrolled situation such as a vascular breach internally, the pathophysiological consequences are numerous. The patient mobilizes fluid and reserve blood volume, notably splenic stored and peripherally circulating erythrocytes, to preferentially supply oxygen to sensitive organs such as the brain and heart. When the global and local delivery of oxygen is insufficient to meet the metabolic needs of the tissues, a cascade of cellular, tissue, and organ dysfunction occurs. If left untreated, the patient dies of hemorrhagic anemic shock. CLINICAL IMPORTANCE: An understanding of the pathophysiological consequences of hemorrhagic shock in horses and their clinical manifestations may help the practitioner understand the severity of blood volume loss, the need for referral, the need for transfusion, and potential outcome. In cases of severe acute uncontrolled hemorrhage, it is essential to recognize the clinical manifestations quickly to best treat the patient, which may include humane euthanasia. KEY POINTS: Uncontrolled hemorrhage may be defined as the development of a vascular breach and hemorrhage that cannot be controlled by interventional hemostasis methods such as external pressure, tourniquet, or ligation. Causes of uncontrolled hemorrhage in horses may be due to non-surgical trauma, surgical trauma, invasive diagnostic procedures including percutaneous organ biopsy, coagulopathy, hypertension, cardiovascular anomaly, vascular damage, neoplasia such as hemangiosarcoma, toxicity, or idiopathic in nature. When a critical volume of blood is lost, the respondent changes in heart rate, splenic blood mobilization, and microcirculatory control can no longer compensate for decreasing oxygen delivery to the tissues In spite of organ-specific microvascular responses (eg, myogenic responses, local mediator modulation of microvasculature, etc), all organs experience decreases in blood flow during severe hypovolemia Acute, fatal hemorrhagic shock is characterized by progressive metabolic acidosis, coagulopathy, and hypothermia, often termed the "triad of death," followed by circulatory collapse.

Abdominocentesis techniques in horses.

BACKGROUND: Abdominocentesis is commonly used to evaluate the abdominal cavity of the horse. This technique provides valuable diagnostic information as well as the means to monitor patients with abdominal diseases being managed medically and to determine their need for surgical management. Complications are uncommon and include trauma to the gastrointestinal tract or spleen, septic peritonitis, or abdominal wall infection. PROCEDURES: This review describes the indications, utility, patient preparation, and instructions for performing abdominocentesis as well as possible complications reported in horses. Step-by-step instructions are provided for the two most commonly used abdominocentesis techniques in horses, which include the use of a needle (18 Ga, 3.8 cm [1.5 in]) and a teat cannula (9.5 cm [3.75 in]). SUMMARY: Peritoneal fluid collection and fluid analysis can be used to confirm diagnosis of intraabdominal pathology including inflammatory, infectious, neoplastic, obstructive, and bowel strangulation, leading to additional diagnostic and therapeutic plans. KEY POINTS: Abdominocentesis is useful as a diagnostic procedure in horses suffering from colic, diarrhea, weight loss, or other conditions involving the abdominal cavity and is an integral component of diagnostic testing for colic at referral institutions or in the field. Abdominal fluid collection using an 18-Ga, 3.8-cm (1.5-in) needle is recommended for adult horses because the needle is long enough to penetrate the peritoneal cavity. The teat cannula technique is recommended for use in adult horses, foals, and miniature horses to reduce the risk of enterocentesis, even though this procedure is more traumatic than using an 18-Ga, 3.8-cm needle. Ultrasonography of the abdomen is a valuable tool in the assessment of any horse with signs of colic, but it is not essential for performing an abdominocentesis successfully.

Collection and administration of blood products in horses: Transfusion indications, materials, methods, complications, donor selection, and blood testing.

BACKGROUND: Blood transfusion is a lifesaving treatment for horses with acute hemorrhage and other causes of anemia. Transfusions improve oxygen delivery to the tissues via increased blood volume and hemoglobin concentration. Certain aspects of equine blood transfusion are challenging, especially in the field situation, and practitioners may be unfamiliar or feel overwhelmed with the process. An understanding of the indications, materials, methods, and techniques as well as donor selection and possible complications will help practitioners successfully implement blood transfusion in clinical practice. PROCEDURES: Blood transfusion involves several steps including appropriate donor selection, cross-matching, blood collection, and administration, as well as monitoring and handling of transfusion reactions. Guidance for each of these steps are detailed in this review. SUMMARY: Blood transfusion is an effective and often lifesaving treatment for managing diseases of blood loss, hemolysis, and decreased RBC production. Equine practitioners require a thorough understanding of the indications for blood transfusion, the immunological principles behind compatibility testing and transfusion reactions, and the technical skills to aseptically collect and administer blood products KEY POINTS: Equine practitioners require a thorough understanding of the indications for blood transfusion, the immunological principles behind compatibility testing and transfusion reactions, and the technical skills to aseptically collect and administer blood products. Because there are over 400,000 possible equine RBC phenotypes, no universal donor exists, and some blood type incompatibilities are likely between any donor and recipient. Therefore, prior to any blood transfusion, donor and recipient blood should be cross-matched Inadequate delivery of oxygen (Do2 ) to the tissues, resulting from low hemoglobin (Hb) concentration, is the most important indication for blood transfusion Neonatal isoerythrolysis most commonly occurs following an anamnestic response in late gestation; it rarely occurs following a primary exposure because the immune response is not strong enough to produce clinically significant alloantibody titers.

Differential effect of isotype on efficacy of anti-tumor necrosis factor alpha chimeric antibodies in experimental septic shock.

Immune complexes containing human gamma (g)1 or murine g2a antibodies generate secondary effector mechanisms via Fc receptor binding or complement activation, whereas those containing human g4 or murine g1 antibodies generally do not. Therefore, isotype selection of therapeutic antibodies may have important clinical consequences. In a rabbit model of human tumor necrosis factor (rhuTNF)-induced pyrexia, a murine/human chimeric g4 anti-human TNF-alpha monoclonal antibody (mAb) (cCB0011) showed a dose-dependent inhibition of pyrexia, whereas a g1 isotype variant of the same mAb gave a marked pyrexia that was seen at all doses indicative of an immune complex-mediated response. To investigate whether isotype difference could influence mAb efficacy in pathological disease states, hamster/murine chimeric g1 and g2a anti-murine TNF-alpha mAbs (TN3g1, TN3g2a) were studied in experimental shock in mice and rats. In lipopolysaccharide-induced shock in mice, treatment with TN3g1 mAb at 30 and 3 mg/kg resulted in 90% survival by 72 h (p < or = 0.004), and prolonged survival to 45 h (p < or = 0.05), respectively, compared with 100% mortality by 27 h in controls. In contrast, a g2a isotype variant of the same mAb (30 mg/kg) resulted in only 10% survival by 72 h (p < or = 0.05). In a neutropenic sepsis model in rats there was greater survival in animals receiving the g1 isotype of TN3 compared with g2a isotype variant (70 vs. 27%; p < or = 0.005) with 100% mortality in the controls. These differences were not due to the pharmacokinetic profiles of the mAbs. In models of experimental shock antibody isotype can affect outcome with inactive isotypes (human g4 and murine g1) being more efficacious than active isotypes (human g1 and murine g2a).

Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27.

The p27 mammalian cell cycle protein is an inhibitor of cyclin-dependent kinases. Both in vivo and in vitro, p27 was found to be degraded by the ubiquitin-proteasome pathway. The human ubiquitin-conjugating enzymes Ubc2 and Ubc3 were specifically involved in the ubiquitination of p27. Compared with proliferating cells, quiescent cells exhibited a smaller amount of p27 ubiquitinating activity, which accounted for the marked increase of p27 half-life measured in these cells. Thus, the abundance of p27 in cells is regulated by degradation. The specific proteolysis of p27 may represent a mechanism for regulating the activity of cyclin-dependent kinases.

A comparison of epidural buprenorphine plus detomidine with morphine plus detomidine in horses undergoing bilateral stifle arthroscopy.

OBJECTIVE: To compare the analgesic efficacy of buprenorphine plus detomidine with that of morphine plus detomidine when administered epidurally in horses undergoing bilateral stifle arthroscopy. STUDY DESIGN: Prospective, randomized, blinded clinical trial. ANIMALS: Twelve healthy adult horses participating in an orthopedic research study. Group M (n = 6) received morphine (0.2 mg kg(-1)) and detomidine (0.15 mg kg(-1)) epidurally; group B (n = 6) received buprenorphine (0.005 mg kg(-1)) and detomidine (0.15 mg kg(-1)) epidurally. METHODS: Horses received one of two epidural treatments following induction of general anesthesia for bilateral stifle arthroscopy. Heart rate (HR), mean arterial blood pressure (MAP), end-tidal CO(2) (Pe'CO(2)), and end-tidal isoflurane concentrations (E'Iso%) were recorded every 15 minutes following epidural administration. Post-operative assessment was performed at 1, 2, 3, 6, 9, 12, and 24 hours after standing; variables recorded included HR, respiratory rate (f(R)), abdominal borborygmi, defecation, and the presence of undesirable side effects. At the same times post-operatively, each horse was videotaped at a walk and subsequently assigned a lameness score (0-4) by three ACVS diplomates blinded to treatment and who followed previously published guidelines. Nonparametric data were analyzed using Wilcoxon's rank-sum test. Inter- and intra-rater agreement were determined using weighted kappa coefficients. Statistical significance was set at p

Arthroscopic removal of palmar/plantar osteochondral fragments from the proximal interphalangeal joint in four horses.

OBJECTIVE: To describe anatomic considerations and arthroscopic technique in horses for arthroscopic removal of palmar/plantar osteochondral fragments from the proximal interphalangeal (PIP) joint. STUDY DESIGN: Retrospective study. ANIMALS: Adult horses (n=4) with osteochondral fragments of the palmar/plantar PIP joint. METHODS: Arthroscopic removal of palmar/plantar osteochondral fragments within the PIP joint was performed with horses in dorsal recumbency under general anesthesia. Medical records of affected horses were reviewed to determine history; physical, lameness, and radiological findings; surgical technique; complications and outcome. RESULTS: Two horses had lameness localized to the PIP joint. Two other horses had lameness suspected, but not confirmed to the pastern region. One of these horses had a history of intermittent lameness, but was not lame on admission. All horses had radiographic evidence of palmar/plantar osteochondral fragmentation within the PIP joint. Fragmentation was located abaxially in 2 horses in the hind limb and axially in 2 horses in the left forelimb. Osteochondral fragments were successfully removed via a palmar/plantar arthroscopic approach in all horses. Three horses returned to previous levels of athletic performance; 1 horse was used for trail riding instead of reining. CONCLUSIONS: Arthroscopy of the palmar/plantar pouch of the PIP joint allowed limited assessment of the joint and removal of osteochondral fragments. CLINICAL RELEVANCE: Arthroscopy of the palmar/plantar PIP joint pouch for assessment and removal of osteochondral fragments is possible and should be considered when lameness is localized to this joint.

Mouse models of cystic fibrosis.

The development of mouse models for cystic fibrosis has provided the opportunity to dissect disease pathogenesis, correlate genotype and phenotype, study disease-modifying genes and develop novel therapeutics. This review discusses the successes and the challenges encountered in characterizing and optimizing these models.

Evolution and stability of chromosomal DNA coamplified with the CAD gene.

We have compared clones of Syrian hamster cells selected for the first amplification of the CAD gene with clones selected for further amplification. The large domain amplified initially was not reamplified as an intact unit. Instead, subregions were reamplified preferentially, and parts of the initial array were often lost. These events reduced the average amount of coamplified DNA accompanying each copy of the selected gene. The degree of amplification was small in the first step (about three extra copies of CAD per cell), but second-step amplifications to a high copy number (up to 60 extra copies per cell) occurred frequently. After several separate steps of amplification, highly condensed arrays that brought many CAD genes close together were formed. In striking contrast to the stability of these highly amplified arrays, the low-copy chromosomal arrays formed early were quite unstable and were often lost completely within 1 or 2 months of growth without selection. The results suggest that different mechanisms may be involved in the first step of amplification and in the later evolution of an already amplified array.

The human UNP locus at 3p21.31 encodes two tissue-selective, cytoplasmic isoforms with deubiquitinating activity that have reduced expression in small cell lung carcinoma cell lines.

The human Unp gene at 3p21.3 has sequence similarity to ubiquitin proteases and has been suggested to play a role in carcinogenesis of the lung (Gray et al., 1995). To investigate this possibility, we isolated cDNAs from several human tissue libraries and found evidence for two major isoforms, encoding proteins predicted to either contain an internal 47 amino acid segment or not. Both are functional in deubiquitination assays, and mutation of a critical conserved cysteine residue to alanine abolished activity. Unp specifies two closely-migrating transcripts whose relative abundance varies among human adult tissues. Antibodies specific to UNP confirm the presence of at least two endogenous protein isoforms of approximately 105-110 kDa in cell lysates, as predicted from the cDNA sequences. Cellular fractionation and immunocytochemistry revealed UNP expression localized primarily in the cytoplasm. When we examined a panel of lung-derived cell lines for both UNP mRNA and protein expression, we found reduced levels of UNP protein in all four small cell lung carcinoma cell lines tested. These findings directly contradict and offer alternative interpretations to a number of previously published observations on Unp.

Antisense targeting of E6AP elevates p53 in HPV-infected cells but not in normal cells.

Invasive cervical cancer is very highly correlated with the presence of high-risk human papillomavirus (HPV) types 16 and 18. Two viral proteins, E6 and E7, act in concert to subvert growth control of infected cells by inactivating the tumor suppressor proteins, p53 and Rb, respectively. E6 is thought to abrogate p53 function by stimulating its degradation via ubiquitin-mediated proteolysis in a reaction requiring E6AP (E6-Associated Protein). Here we evaluate the in vivo role of E6AP in p53 degradation in normal and HPV-infected cell types using antisense phosphorothioate oligodeoxynucleotides (S-ODNs). This study shows that reduction of E6AP in vivo in high-risk HPV-infected cells leads to an elevation of p53, confirming the function of E6AP predicted by in vitro experiments. Further, we demonstrate that reduction of E6AP in normal cells has no effect on p53 levels, indicative of an E6AP-indpendent mechanism for p53 degradation. These experiments show that inhibition of intermediate proteins in the ubiquitin-mediated proteolysis pathway (ubiquitin-conjugating enzymes or associated recognition proteins) can result in specific inhibition of substrate degradation. We propose that modulation of p53 levels by elimination of E6AP function may have therapeutic potential for cervical cancer.

SCF(beta-TRCP) and phosphorylation dependent ubiquitinationof I kappa B alpha catalyzed by Ubc3 and Ubc4.

NF kappa B is an important transcriptional regulator of multiple pro-inflammatory genes. In non-stimulated cells NF kappa B is anchored in the cytoplasm via the inhibitory protein I kappa B alpha. Following exposure to diverse pro-inflammatory signals (e.g. TNF alpha, IL1, LPS) various signal transduction cascades are initiated converging on the I kappa B kinase (IKK). IKK phosphorylates I kappa B alpha on serines 32 and 36 signaling the inhibitory protein for ubiquitin-mediated degradation. The SCF beta-TRCP complex is the ubiquitin ligase responsible for mediating phosphorylation dependent ubiquitination of I kappa B alpha. Here we reconstitute phosphorylation dependent ubiquitination of I kappa B alpha using recombinant components. Our results suggest that the cullin specificity of the SCF complex may reflect its ability to associate with Rbx1. We demonstrate specific ubiquitination of I kappa B alpha by Ubc3 and Ubc4 in a phosphorylation and SCF beta-TRCP dependent manner and that both are capable of associating with the SCF beta-TRCP complex isolated from human cells. Finally, we show that Ubc4 is in excess to Ubc3 in THP.1 cells and 19 times more efficient in catalyzing the reaction, suggesting that Ubc4 is the preferentially used Ubc in this reaction in vivo. Our results also suggest that ubiquitin is transferred directly from the Ubc to phospho-I kappa B alpha in a SCF beta-TRCP dependent reaction. Oncogene (2000) 19, 3529 - 3536

The ubiquitin-mediated proteolytic pathway as a therapeutic area.

Ubiquitin-mediated proteolysis is involved in the turnover of many short-lived regulatory proteins. This pathway leads to the covalent attachment of one or more multiubiquitin chains to target substrates which are then degraded by the 26S multicatalytic proteasome complex. Multiple classes of regulatory enzymes have been identified that mediate either ubiquitin conjugation or ubiquitin deconjugation from target substrates. Timed destruction of cellular regulators by the ubiquitin-proteasome pathway plays a critical role in ensuring normal cellular processes. This review provides multiple examples of key growth regulatory proteins whose levels are regulated by ubiquitin-mediated proteolysis. Pharmacological intervention which alters the half-lives of these cellular proteins may have wide therapeutic potential. Specifically, prevention of p53 ubiquitination (and subsequent degradation) in human papilloma virus positive tumors, and perhaps all tumors retaining wild-type p53 but lacking the retinoblastoma gene function, should lead to programmed cell death. Specific inhibitors of p27 and cyclin B ubiquitination are predicted to be potent antiproliferative agents. Inhibitors of IkappaB ubiquitination should prevent NFkappaB activation and may have utility in a variety of autoimmune and inflammatory conditions. Finally, we present a case for deubiquitination enzymes as novel, potential drug targets.

Does skin-to-skin contact and breast feeding at birth affect the rate of primary postpartum haemorrhage: Results of a cohort study.

OBJECTIVE: to examine the effect of skin-to-skin contact and breast feeding within 30 minutes of birth, on the rate of primary postpartum haemorrhage (PPH) in a sample of women who were at mixed-risk of PPH. DESIGN: retrospective cohort study. SETTING: two obstetric units plus a freestanding birth centre in New South Wales (NSW) Australia. PARTICIPANTS: after excluding women (n=3671) who did not have opportunity for skin to skin and breast feeding, I analysed birth records (n=7548) for the calendar years 2009 and 2010. Records were accessed via the electronic data base ObstetriX. INTERVENTION: skin to skin contact and breast feeding within 30 minutes of birth. MEASURES: outcome measure was PPH i.e. blood loss of 500ml or more estimated at birth. Data was analysed using descriptive statistics and logistic regression (unadjusted and adjusted). FINDINGS: after adjustment for covariates, women who did not have skin to skin and breast feeding were almost twice as likely to have a PPH compared to women who had both skin to skin contact and breast feeding (aOR 0.55, 95% CI 0.41-0.72, p<0.001). This apparently protective effect of skin to skin and breast feeding on PPH held true in sub-analyses for both women at 'lower' (OR 0.22, 95% CI 0.17-0.30, p<0.001) and 'higher' risk (OR 0.37 95% CI 0.24-0.57), p<0.001. KEY CONCLUSIONS AND IMPLICATION FOR PRACTICE: this study suggests that skin to skin contact and breastfeeding immediately after birth may be effective in reducing PPH rates for women at any level of risk of PPH. The greatest effect was for women at lower risk of PPH. The explanation is that pronurturance promotes endogenous oxytocin release. Childbearing women should be educated and supported to have pronurturance during third and fourth stages of labour.