Significance of immunohematologic testing in mother and newborn ABO incompatibility.

The aim of this study was to define risk factors for jaundice and anemia in newborns with a positive direct antiglobulin test (DAT) and/or with an incompatible crossmatch due to ABO incompatibility between mother and newborn. ABO incompatibility has become a more significant cause of hemolytic disease of the fetus and newborn since the introduction of effective anti-D prophylaxis. The condition is common and, if clinically significant at all, causes only mild jaundice, which can be treated with phototherapy (PT). However, rare and serious presentations, requiring transfusion therapy, have been noted. Clinical, laboratory, and immunohematologic data were collected retrospectively from medical records of ABO-incompatible newborns and their mothers over a 5-year period (2016-2020) from University Hospital Centre Zagreb. Two groups of newborns were compared: those who needed medical intervention because of hyperbilirubinemia or anemia and those who did not. Within the group of newborns requiring intervention, we also compared those with A and B blood groups. Over the 5-year period, 72 of 184 (39%) newborns required treatment. The treatment was PT in 71 (38%) newborns and erythrocyte transfusion in 2 (1%). In 112 (61%) newborns, ABO incompatibility was an accidental finding while performing blood group typing; these newborns did not require any therapy. In conclusion, we found a statistical, but not clinically significant, difference between the groups of treated and untreated newborns, related to the mode of delivery and DAT positivity within hours of delivery. There were no statistically significant differences in characteristics between the groups of treated newborns, except for two newborns with blood group A who received erythrocyte transfusions.

Osmolality and solute composition are strong regulators of AQP2 expression in renal principal cells.

The water permeability of the renal collecting duct is regulated by the insertion of aquaporin-2 (AQP2) into the apical plasma membrane of epithelial (principal) cells. Using primary cultured epithelial cells from the inner medulla of rat kidney (IMCD cells), we show that osmolality and solute composition are potent regulators of AQP2 mRNA and protein synthesis, as well as the classical cAMP-dependent pathway, but do not affect the arginine vasopressin-induced AQP2 shuttle. In the presence of the cAMP analog dibutyryl cAMP (DBcAMP, 500 microM), NaCl and sorbitol, but not urea, evoked a robust increase of AQP2 expression in IMCD cells, with NaCl being far more potent than sorbitol. cAMP-responsive element-binding protein phosphorylation increased with DBcAMP concentrations but was not altered by changes in osmolality. In the rat and human AQP2 promoter, we identified a putative tonicity-responsive element. We conclude that, in addition to the arginine vasopressin/cAMP-signaling cascade, a further pathway activated by elevated effective osmolality (tonicity) is crucial for the expression of AQP2 in IMCD cells, and we suggest that the effect is mediated via the tonicity-responsive element.

Rho inhibits cAMP-induced translocation of aquaporin-2 into the apical membrane of renal cells.

First published August 8, 2001; 10.1152/ajprenal.00091.2001.-We have recently demonstrated that actin depolymerization is a prerequisite for cAMP-dependent translocation of the water channel aquaporin-2 (AQP2) into the apical membrane in AQP2-transfected renal CD8 cells (29). The Rho family of small GTPases, including Cdc42, Rac, and Rho, regulates the actin cytoskeleton. In AQP2-transfected CD8 cells, inhibition of Rho GTPases with Clostridium difficile toxin B or with C. limosum C3 fusion toxin, as well as incubation with the Rho kinase inhibitor, Y-27632, caused actin depolymerization and translocation of AQP2 in the absence of the cAMP-elevating agent forskolin. Both forskolin and C3 fusion toxin-induced AQP2 translocation were associated with a similar increase in the osmotic water permeability coefficient. Expression of constitutively active RhoA induced formation of stress fibers and abolished AQP2 translocation in response to forskolin. Cytochalasin D induced both depolymerization of F-actin and AQP2 translocation, suggesting that depolymerization of F-actin is sufficient to induce AQP2 translocation. Together, these data indicate that Rho inhibits cAMP-dependent translocation of AQP2 into the apical membrane of renal principal cells by controlling the organization of the actin cytoskeleton.

Ht31: the first protein kinase A anchoring protein to integrate protein kinase A and Rho signaling.

In an attempt to isolate protein kinase A anchoring proteins (AKAPs) involved in vasopressin-mediated water reabsorbtion, the complete sequence of the human AKAP Ht31 was determined and a partial cDNA of its rat orthologue (Rt31) was cloned. The Ht31 cDNA includes the estrogen receptor cofactor Brx and the RhoA GDP/GTP exchange factor proto-lymphoid blast crisis (Lbc) sequences. The Ht31 gene was assigned to chromosome 15 (region q24-q25). It encodes Ht31 and the smaller splice variants Brx and proto-Lbc. A protein of the predicted size of Ht31 (309 kDa) was detected in human mammary carcinoma and HeLa cells. Anti-Ht31/Rt31 antibodies immunoprecipitated RhoA from primary cultured rat renal inner medullary collecting duct cells, indicating an interaction between the AKAP and RhoA in vivo. These results suggest that Ht31/Rt31 represent a new type of AKAP, containing both an anchoring and a catalytic domain, which appears to be capable of modulating the activity of an interacting partner. Ht31/Rt31 have the potential to integrate Rho and protein kinase A signaling pathways, and thus, are prime candidates to regulate vasopressin-mediated water reabsorbtion.

Cell volume kinetics of adherent epithelial cells measured by laser scanning reflection microscopy: determination of water permeability changes of renal principal cells.

The water channel aquaporin-2 (AQP2), a key component of the antidiuretic machinery in the kidney, is rapidly regulated by the antidiuretic hormone vasopressin. The hormone exerts its action by inducing a translocation of AQP2 from intracellular vesicles to the cell membrane. This step requires the elevation of intracellular cyclic AMP. We describe here a new method, laser scanning reflection microscopy (LSRM), suitable for determining cellular osmotic water permeability coefficient changes in primary cultured inner medullary collecting duct (IMCD) cells. The recording of vertical-reflection-mode x-z-scan section areas of unstained, living IMCD cells proved useful and valid for the investigation of osmotic water permeability changes. The time-dependent increases of reflection-mode x-z-scan section areas of swelling cells were fitted to a single-exponential equation. The analysis of the time constants of these processes indicates a twofold increase in osmotic water permeability of IMCD cells after treatment of the cells both with forskolin, a cyclic AMP-elevating agent, and with Clostridium difficile toxin B, an inhibitor of Rho proteins that leads to depolymerization of F-actin-containing stress fibers. This indicates that both agents lead to the functional insertion of AQP2 into the cell membrane. Thus, we have established a new functional assay for the study of the regulation of the water permeability at the cellular level.

An inhibitory role of Rho in the vasopressin-mediated translocation of aquaporin-2 into cell membranes of renal principal cells.

Vasopressin regulates water reabsorption in renal collecting duct principal cells by a cAMP-dependent translocation of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the cell membrane. In the present work primary cultured inner medullary collecting duct cells were used to study the role of the proteins of the Rho family in the translocation of AQP2. Clostridium difficile toxin B, which inhibits all members of the Rho family, Clostridium limosum C3 toxin, which inactivates only Rho, and the Rho kinase inhibitor, Y-27632, induced both depolymerization of actin stress fibers and AQP2 translocation in the absence of vasopressin. The data suggest an inhibitory role of Rho in this process, whereby constitutive membrane localization is prevented in resting cells. Expression of constitutively active RhoA induced formation of actin stress fibers and abolished AQP2 translocation in response to elevation of intracellular cAMP, confirming the inhibitory role of Rho. Cytochalasin D induced both depolymerization of the F-actin cytoskeleton and AQP2 translocation, indicating that depolymerization of F-actin is sufficient to induce AQP2 translocation. Thus Rho is likely to control the intracellular localization of AQP2 via regulation of the F-actin cytoskeleton.

The mechanisms of aquaporin control in the renal collecting duct.

The antidiuretic hormone arginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells. Central to its antidiuretic action in mammals is the exocytotic insertion of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the apical membrane of principal cells, an event initiated by an increase in cAMP and activation of protein kinase A. Water is then reabsorbed from the hypotonic urine of the collecting duct. The water channels aquaporin-3 (AQP3) and aquaporin-4 (AQP4), which are constitutively present in the basolateral membrane, allow the exit of water from the cell into the hypertonic interstitium. Withdrawal of the hormone leads to endocytotic retrieval of AQP2 from the cell membrane. The hormone-induced rapid redistribution between the interior of the cell and the cell membrane establishes the basis for the short term regulation of water permeability. In addition water channels (AQP2 and 3) of principal cells are regulated at the level of expression (long term regulation). This review summarizes the current knowledge on the molecular mechanisms underlying the short and long term regulation of water channels in principal cells. In the first part special emphasis is placed on the proteins involved in short term regulation of AQP2 (SNARE proteins, Rab proteins, cytoskeletal proteins, G proteins, protein kinase A anchoring proteins and endocytotic proteins). In the second part, physiological and pathophysiological stimuli determining the long term regulation are discussed.

Protein kinase A anchoring proteins are required for vasopressin-mediated translocation of aquaporin-2 into cell membranes of renal principal cells.

The antidiuretic hormone arginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells by inducing a cAMP-dependent translocation of water channels (aquaporin-2, AQP-2) from intracellular vesicles into the apical cell membranes. In subcellular fractions from primary cultured rat inner medullary collecting duct (IMCD) cells, enriched for intracellular AQP-2-bearing vesicles, catalytic protein kinase A (PKA) subunits and several protein kinase A anchoring proteins (AKAPs) were detected. In nonstimulated IMCD cells the majority of AQP-2 staining was detected intracellularly but became mainly localized within the cell membrane after stimulation with AVP or forskolin. Quantitative analysis revealed that preincubation of the cells with the synthetic peptide S-Ht31, which prevents the binding between AKAPs and regulatory subunits of PKA, strongly inhibited AQP-2 translocation in response to forskolin. Preincubation of the cells with the PKA inhibitor H89 prior to forskolin stimulation abolished AQP-2 translocation. In contrast to H89, S-Ht31 did not affect the catalytic activity of PKA. These data demonstrate that not only the activity of PKA, but also its tethering to subcellular compartments, are prerequisites for cAMP-dependent AQP-2 translocation.

The influence of increased intra-abdominal pressure on blood coagulation values.

AIMS/BACKGROUND: Increased intra-abdominal pressure during laparoscopic cholecystectomy changes pressure values in the thoracic cavity, in blood vessels, and in biochemical blood values. METHODOLOGY: In this prospective study, we investigate whether CO2 pneumoperitoneum causes blood absorption and whether there is an increased risk of thromboembolism because of its mechanical influence on reographic and biochemical blood attributes. We analyzed pH, BE and arterial blood pCO2 pre-operatively, three times during surgery and post-operatively; and blood coagulation values pre-, during and 30 minutes after the operation. Blood samples were taken from 75 patients, divided into two groups. The first group consisted of 50 patients on whom laparoscopic cholecystectomies were performed, while the second (control) group consisted of 25 patients who were operated on by open laparotomy. All patients had a pre-operative physical status of ASA I and ASA II. RESULTS: There was no statistically significant difference in pCO2 in any measurement, but in the group of patients treated with a laparoscopy there were statistically significant increases in pCO2 in three measurements during the operation. CONCLUSIONS: The data show that during the laparoscopy the pneumoperitoneum had no physical or biochemical influence on coagulation values and so reduced the risk of thromboembolism.

Aquaporin-2 expression in primary cultured rat inner medullary collecting duct cells.

Cultured renal epithelial cells rapidly downregulate expression of the vasopressin-regulated water channel aquaporin-2 (AQP-2). Our aim was to define conditions that favor maintenance of AQP-2 expression in vitro without genetic manipulation. We show here that primary cultures of rat inner medullary collecting duct (IMCD) cells retain AQP-2 expression for at least 6 days when grown with dibutyryl cAMP (DBcAMP) supplementation. We also found that coating the culture dishes with type IV collagen, rather than rat-tail collagen, retards AQP-2 downregulation. Immunofluorescence and biochemical studies indicate a shuttling of AQP-2-bearing vesicles after stimulation with vasopressin or forskolin. Rab3 proteins, known to be involved in regulated exocytosis, were detected only in cells grown in the presence of DBcAMP. Using the adenylyl cyclase assay, we confirmed the functional integrity of the vasopressin V2 receptor in a broken cell preparation. Our data show that cAMP supplementation is sufficient for the maintenance of AQP-2 expression in primary cultured cells. The model system established here allows the study of the regulation of genes encoding the antidiuretic machinery at the cellular level.

Meaning of prognostic factors in patients with acute pancreatitis.

The aim of the study was to investigate reliability of Ranson's prognostic factors in our 43 patients with acute pancreatitis during a ten year period (1984-1993). Based on the retrospective analysis and Ranson's prognostic factors, the patients were divided into groups and severe form of pancreatitis. The patients with less than 3 prognostic factors were classified into the mild form group. The patients who had 3 or more Ranson's factors were considered as having the severe form of the disease. Sixty percent of the patients with severe form of the disease survived. All the patients who died (2 females and 7 males) had more than 6 Ranson's prognostic factors and died within 15 days from admission to hospital. Of the patients who died, eight had been operated on for multiorgan injury. None of the patients with mild form of the disease died. The prognosis of the outcome of pancreatitis with severe complications and of death mode, on the basis of Ranson's factors, was found to be more certain when more factors were present.

Surgical resection in the treatment of bulbostenosis.

The aim of the study was to evaluate surgical methods used in the treatment of gastric outlet obstruction. From 1983 till 1994, 151 patients were surgically treated for gastric outlet stenosis at the Dubrava University Hospital. There were 120 males and 31 females, mean age 54.13 +/- 13.07 years. The treatment was evaluated by a modified Visick index. The most commonly surgical method used was Billroth I gastric resection. Postoperative complications developed in 8% of the patients, while early postoperative mortality was 2%. The authors compiled data on the postoperative course for 46 patients, 20 of whom were examined gastroscopically and radiologically. One recurrence which required reoperation was verified. Ninety-four percent of these patients were evaluated as Visick I and II. Billroth I gastric resection yields good results in treating gastric outlet obstruction, but postoperative complications and early postoperative mortality of 2% make it less suitable than proximal selective vagotomy with dilatation or duodenoplasty.

Anaphylactic shock in a female patient due to a spontaneous rupture of a hepatic hydatid cyst: a case report.

A 27-year-old female patient, a refugee from Bosnia and Herzegovina, was admitted to our hospital in a state of shock. She developed an anaphylactic reaction. The US and CT of abdomen revealed a spontaneous rupture of a hepatic hydatid cyst. An emergency operative procedure was performed and in the postoperative period she was treated conservatively with albendazol during the next two months. There was no recurrence of the disease.

Three-dimensional observation of wound temperature in primary healing.

In a group of 30 patients the temperature in the epigastric region was followed pre-operatively for four days. After upper medial laparotomy the temperature of the wound and its surroundings was followed during eight post-operative days. Thus every examinee was a control to himself. The temperature was measured on AGA 780 thermovision system. The advantages of thermovision in observing temperature in primary healing are manifold. The method is painless, non-contact, thereby decreasing the risk of contamination from outside, gives an absolutely accurate image of temperature over a larger or smaller area, and analysis of the thermogram is relatively simple. The defining of the healing process as thermal change in intensity, extensiveness and time is, in essence, three-dimensional. It gives the possibility of accurate indirect observation of biochemical and chemico-physical changes in the wound and its surroundings.

A prospective study of the application of transfusions in a field hospital during the war in Croatia.

Use of blood products are recommended worldwide as a substitution for whole blood. In the case of war wounds, because of their specificity, this is not possible. We studied prospectively the use of blood and blood products during management of the wounded in a field hospital during the war in Croatia. During a 6-month period, 178 soldiers out of a total number of 593 were hospitalized, and of this number 64 (36%) received transfusions. Localization of the wound, and speed and quality of first aid had significant effects on the average amount of blood used. Consequently, norms for blood use in wartime in west European countries and NATO are lower than what we used for each wounded patient. Ensuring an adequate amount of whole blood still remains one of the primary tasks of the transfusion service in wartime.

Contact Thermography in a study of primary healing of surgical wounds.

Occurrences in the wound until complete healing are manifested in different ways, one of which is temperature change. The aim of this study was establish if there is regularity in temperature changes in wounds healing by primary intention. Thirty examinees (27 female, 3 male) admitted for gallstone surgery were included. Temperature was measured for eight post-operative days at a fixed time each day and in the same room using the semi-quantitative, contact method of liquid crystal strips. The findings demonstrated that there were few differences among the collective thermal images of individual examines for any given post-operative day. During the first 3 post-operative days, the temperatures rose, with few differences between the temperatures of the wounds and their wider surroundings. From day 4 through 8, the temperatures of the wounds and surroundings fell gradually. Stitches were removed on day 7, and on day 8 only the narrow zones of the incision sites were warmer than the surroundings. The findings suggest that there is regularity in the course of temperature of the surgical wound in primary healing. The clinical implications are that the persistence of a wider zone of increased temperature after day 4 predicts the possibility of wound infection and disturbed healing.

[The temperature dynamics during the healing processing of a surgical wound]. / Die Temperaturdynamik während des Heilungsprozesses der chirurgischen Wunde.

Using biomechanical principles, the qualitative occurrences in the wound were quantified. On the basis of the empirical knowledge that the wound, from its inception to healing, is warmer than its surroundings, we presumed that there was a certain regularity in the course of temperature values of the wound during primary healing. For measuring the temperature of the wound and its surroundings an electric thermometer of the ELLAB type was used. First, the temperature of the skin in the epigastric region in 30 healthy examinees, who were due to undergo a gall-bladder concrements operation was determined. The temperatures were measured within a traced dotted network. After the operation, in the same examinees, the temperatures were registered proximal to the wound and in its closer surroundings throughout the first eight post-operative days. Statistically evaluated the results showed that the temperature of the wound and of its surroundings, measured after operation, was higher than prior to operation. The temperature of the wound gradually rises up to the third post-operative day, and then gradually falls from the fourth post-operative day. This course of temperature is explained in part by the biological occurrences in the wound. The inflammatory reaction as a consequence of damage to the tissue is responsible for the initial increase of wound temperature. It lasts a limited time and has a limited intensity. This could be proved by histological preparations of the wound. The persistence of increased temperature after the third post-operative day is a sign of disturbed healing and predicts infection and secondary healing.(ABSTRACT TRUNCATED AT 250 WORDS)

[The effect of atmospheric conditions on the occurrence of peptic ulcer hemorrhage]. / Utjecaj atmosferskih uvjeta na ucestalost krvarenja iz peptickog ulkusa.

The influence of atmospheric factors on the frequency of bleeding from the peptic ulcer was studied within the period from April 1, 1984, to March 31, 1989, consequently through 1826 days. The average daily atmospheric pressure, the average daily temperature and the relative humidity have been examined. There were 1102 cases of bleeding peptic ulcer, 537 bleeding gastric ulcers and 565 bleeding duodenal ulcers. During the study period there were 454 days with bleeding form ventricular ulcer and 465 days with bleeding from duodenal ulcer. There was 793 days with bleeding form either lesion. The discriminatory analysis demonstrated that the atmospheric pressure is the variable that discriminates the days with bleeding and the days prior to bleeding from the days without bleeding. The relative humidity occurs as the relevant discriminatory variable in the days prior to bleeding for the duodenal ulcer group and for the entire group. The centroids of the discriminatory function demonstrate that the days with ulcer bleeding are characterized by the fall of atmospheric pressure. The factor analysis of meterological variables clearly shows the correlation of the atmospheric pressure and the bleeding regardless to the localisation of bleeding ulcer, where the greatest number of bleedings is correlated with lower atmospheric pressure. We conclude that the incidence of bleeding form the peptic ulcer of the stomach and duodenum correlates in great measure with low atmospheric pressure in the days prior to bleeding and in the days of bleeding, as well as with fall of atmospheric pressure in the days of bleeding with respect to previous day.(ABSTRACT TRUNCATED AT 250 WORDS)