Potential of Ocimum basilicum L. and Salvia officinalis L. essential oils against biofilms of P. aeruginosa clinical isolates.

Biofilms are complex communities of microorganisms, responsible for more than 60% of the chronic human infections and they represent one of the leading concerns in medicine. Pseudomonas aeruginosa is human pathogenic bacteria which causes numerous diseases and is known for its ability to produce biofilm. Ocimum basilicum L. (basil) and Salvia officinalis L. (sage) are widely used plants in traditional medicine for the treatment of different conditions. Therefore, the aim of this study was to investigate the potential of basil and sage essential oils against P. aeruginosa biofilm producing strains. The efficacy of two essential oils on P. aeruginosa biofilm forming ability was determined using crystal violet method. Out of 15 strains isolated from different clinical biological samples, two were strong, 11 moderate and one weak biofilm producer. Good efficacy of sage essential oil towards strong and weak biofilm producers, but not of basil essential oil, was observed. In the case of moderate biofilm producers, 81.8% showed lower biofilm production after incubation with the sage oil, while 63.6% showed the reduction of biofilm production after basil essential oil treatment. The obtained results showed high potential of both oils for the treatment of persistent infections caused by Pseudomonas aeruginosa biofilms.

Control of human and plant fungal pathogens using pentaene macrolide 32, 33-didehydroroflamycoin.

AIMS: The aim of this study was to address the toxicity of recently described polyene macrolide 32, 33-didehydroroflamycoin (DDHR) on a wide range of fungal pathogens and its potential to control plant fungal diseases. METHODS AND RESULTS: The antifungal activity of DDHR in vitro was examined against common human and plant pathogenic fungi using a broth microdilution assay and a disk diffusion assay. Minimum inhibitory concentrations ranged from 12·5 to 35 µg ml(-1) . A radial growth inhibition assay showed that DDHR inhibited mycelia growth, inducing mycelial necrosis and affecting sporulation. During the in vivo assay on apple fruits administration of DDHR 1 h before fungal inoculation inhibited spreading of the infection. Importantly, DDHR exhibited no phytotoxic effects on the model plant, Capsicum annum, verified by the plant growth rate and chlorophyll content. CONCLUSIONS: DDHR inhibits growth of various plant pathogens in vitro with the strongest activity against Alternaria alternata, Colletotrichum acutatum and Penicillium expansum, and protects apple fruits from decay. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the inhibitory effect of DDHR on important pathogenic fungal isolates. DDHR could be a good scaffold for developing new antifungal agents for fruit and vegetable protection.

Didehydroroflamycoin pentaene macrolide family from Streptomyces durmitorensis MS405(T) : production optimization and antimicrobial activity.

AIMS: The aim of this study was to improve production of pentaene 32,33-didehydroroflamycoin (DDHR) in Streptomyces durmitorensis MS405 strain to obtain quantities sufficient for in depth analysis of antimicrobial properties. METHODS AND RESULTS: Through classical medium optimization conditions for stable growth, DDHR production within 7 days of incubation was established. Yields of 215 mg l(-1) were achieved in shake flask experiments in complex medium with mannitol as the primary carbon source. DDHR had poor antibacterial activity with minimal inhibitory concentrations (MIC) of 400 µg ml(-1) for Staphylococcus aureus and Bacillus subtilis, while MIC of 70 µg ml(-1) was determined for Candida albicans. Using flow cytometry and fluorescent microscopy, it was demonstrated that DDHR induced membrane damage in C. albicans followed by cell death. Combination studies with known antifungal nystatin showed that DDHR is a promising agent for the development of novel antimycotic treatments potentially less toxic for human cells. CONCLUSIONS: Pentaene didehydroroflamycoin has no antibacterial activity but can be further developed for the application in antifungal therapy. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report on the stable and production in high yields of a novel pentaene family that acts on Candida cell membranes and can be used in combination with known antifungals. Polyenes are still antifungal antibiotics of choice, and therefore, isolation and production of new lead structures are highly significant.

Viscum album [L.] extract therapy in patients with locally advanced or metastatic pancreatic cancer: a randomised clinical trial on overall survival.

BACKGROUND: The unfavourable side-effects of late-stage pancreatic cancer treatments call for non-toxic and effective therapeutic approaches. We compared the overall survival (OS) of patients receiving an extract of Viscum album [L.] (VaL) or no antineoplastic therapy. METHODS: This is a prospective, parallel, open label, monocentre, group-sequential, randomised phase III study. Patients with locally advanced or metastatic cancer of the pancreas were stratified according to a binary prognosis index, composed of tumour stage, age and performance status; and were evenly randomised to subcutaneous injections of VaL extracts or no antineoplastic therapy (control). VaL was applied in a dose-escalating manner from 0.01 mg up to 10mg three times per week. Patients in both groups received best supportive care. The primary end-point was 12-month OS, assessed in a group-sequential analysis. FINDINGS: We present the first interim analysis, including data from 220 patients. Baseline characteristics were well balanced between the study arms. Median OS was 4.8 for VaL and 2.7 months for control patients (prognosis-adjusted hazard ratio, HR=0.49; p<0.0001). Within the 'good' prognosis subgroup, median OS was 6.6 versus 3.2 months (HR=0.43; p<0.0001), within the 'poor' prognosis subgroup, it was 3.4 versus 2.0 months respectively (HR=0.55; p=0.0031). No VaL-related adverse events were observed. CONCLUSION: VaL therapy showed a significant and clinically relevant prolongation of OS. The study findings suggest VaL to be a non-toxic and effective second-line therapy that offers a prolongation of OS as well as less disease-related symptoms for patients with locally advanced or metastatic pancreatic cancer.

[Changes in concentrations of sera proteins in war wound].

UNLABELLED: During the general reaction to trauma, substantional changes in protein composition of sera occure. The aim of the prospective study was to investigate net change in total protein and albumin concentrations, as well as albumin/globulin ratio in sera of war casualties during the first 14 posttraumatic days, and to establish the correlation between these changes and severity of trauma. Subjects were 79 war casualties. CONTROLS: 33 blood donors. METHODS: Injury severity was determined according to ISS and blood samples were collected 12 hours after trauma, then on the 1st, 2nd, 5th and 14th posttraumatic day. In war casualties values of total protein and albumin concentrations and albumin/globulin ratio were significantly decreased. Minimal concentrations were measured on 2nd posttraumatic day (589.04 g/l for total proteins, p; 36.66.21 g/l for albumins, p) or on 5th day (0.860.2 for albumin/globulin ratio, p). CONCLUSIONS: During the acute-phase response to trauma, significant changes in concentration of total proteins, albumins and albumin/globulin ratio occure in sera of war casualties. These changes are the most promminent during the first 5 days, with tendency for normalization after that. Intensity of these changes depends of the severity of trauma.

[C-reactive protein as an indicator of the severity of war injuries]. / C-reaktivni protein kao pokazatelj tezine ratne povrede.

UNLABELLED: During the general reaction to trauma, acute phase proteins are synthetized. The aim of the prospective study was to determine CRP concentrations in sera of war casualties during the first 14 posttraumatic days, and to establish the correlation between these changes and severity of trauma. Subjects were 79 war casualties. CONTROLS: 33 blood donors. METHODS: Injury severity was determined according to ISS and CRP concentrations with immunonephelometric analysis. Blood samples were collected 12 hours after trauma, then on the 1st, 2nd, 5th and 14th posttraumatic day. In war casualties CRP values were significantly increased (56.257.53 mg/dl after 12 hrs, 107.0976.08 on 1st, 144.3570.23 on 2nd, 71.42558.66 on 5th and 37.656.14 on 14th posttraumatic day; p). Significant differences were observed between groups with ISS and ISS12 (p) in first two days and later between group with ISS24 (144.1766.94 mg/dl on 5th and 111.588.5 on 14th posttraumatic day) and others (p). CONCLUSIONS: During the acute-phase response to trauma, significant changes in concentration of CRP occur in sera of war casualties. These changes are the most prominent during the first 48 hours, with tendency for normalization after the 5th day. Intensity of these changes depend on the severity of trauma. CRP is valid marker of war wound severity.

[Correlation of size of the primary tumor and axillary node status with the p53 tumor suppressor gene in carcinoma of the breast]. / Korelacija velicine primarnog tumora i aksilarnog nodusnog statusa sa tumor supresornim genom p53 kod karcinoma dojke.

Correlation of standard pathomorphological prognostic parameters, primary tumor size and axillary nodal status with new prognostic factor in breast carcinoma: tumor suppressor gene p53 was analyzed. The studied sample included 65 women who underwent surgery for breast carcinoma at the Surgical Clinic of Clinical Center Banja Luka, from January 1st 1997 till January 1st 1999. Statistical data analysis was performed and correlation of prognostic factors was determined. The majority of authors in this field agree that the primary tumor size and axillary nodal status are the two most important prognostic factors. These factors are the best predictors of prognosis and survival of women who had the tumor and were operated on. Tumor markers were immunohistochemically determined in the last ten years and, according to the majority of authors, are still considered the additional or relative prognostic factors in breast carcinoma. Their prognostic value and significance increase almost daily. Most frequently determined tumor markers are bcl-2, pS2, Ki-67 and p53. There was a positive, directly proportional relationship between primary tumor size and tumor suppressor gene p53, but there was no positive correlation between the axillary nodal status and tumor suppressor gene p53. Significance of determination of new tumor markers as the prognostic factors was emphasized. These markers represent a powerful tool in the early detection and prevention of breast carcinoma.

Increased occurrence of PGP 9.5-immunoreactive epidermal Langerhans cells in rat plantar skin after sciatic nerve injury.

This study examines the occurrence and distribution of epidermal dendritic cells (DCs) in cryostate sections from plantar skin in normal rats and in rats with a crush injury or neurotomy and suture of the sciatic nerve. The dendritic cells were visualized with antibodies against protein-gene product 9.5 (PGP 9.5). Counts under the fluorescence microscope showed that the occurrence of dendritic cells is increased and that the proportion of dendritic cells in the basal layer is elevated 3 months after sciatic neurotomy and suture but not after a crush lesion. The countings also revealed that the number of cells is elevated as soon as 1 week after neurotomy and suture. Labelling with specific antibodies showed that the dendritic cells examined represent Langerhans cells (LCs). These observations show that there is a neural influence on the occurrence and distribution of PGP 9.5-immunoreactive epidermal Langerhans cells. Whether this influence is direct or indirect remains to be ascertained.

Indirect effect of sciatic nerve injury on the epidermal thickness of plantar glabrous skin in rats.

In patients with nerve injuries, the skin over the denervated part tends to atrophy. Few experimental studies have dealt with this issue. In this paper we examine the effects of nerve injury on the epidermis in plantar glabrous skin in rats. Analysis of paraffin sections showed that the epidermis becomes abnormally thin after sciatic neurotomy and suture or neurectomy, but not after nerve crush. Autoradiographic analysis showed a subnormal number of thymidine-labelled epidermal cells in plantar skin of rats subjected to sciatic neurectomy. However, after selective division of the sciatic foot branches, with preserved function of thigh and leg muscles, the number of labelled epidermal cells did not differ significantly between the side operated on and the control side. We conclude that the effect of sciatic nerve division on plantar skin in rats is caused by the motor deficiency in the foot rather than by deficient innervation of the skin.

[Necrosis of the omental flap]. / Nekroze reznja omentuma.

The aim of this research is to review the frequency of the omental flap necrosis comparing the vascularized omental flaps based on the left or right gastroepiploic vessels. The first 100 patients, with injuries of spinal cord on different levels, are included in this prospective clinical study with follow-up from 12 to 24 months. The special surgical technique was used for preparation of omental pedicled graft, for its lengthening and transposition to the level of the spinal cord injury and direct and indirect signs of the omental flap necrosis were studied. In our patients there was no necrosis of the omental grafts based on the left gastroepiploic artery. The insufficiency of the left gastroepiploic artery was not present in any patient and so it was not the reason of the omental flap necrosis. Devascularisation of the great gastric curvature until to the root of the left gastroepiploic artery, administration of the prophylactic doses of the Heparin and to put gently pressure on the omental flap do not contribute to the appearance of the omental flap necrosis. Based on our experiences and on results of this research we conclude that this way of forming the omental graft can be used for the other omentopexies.

[Left-sided gallbladder and hypoplasia of the right hepatic lobe--a rare congenital anomaly]. / Levostrana zucna kesa i hipoplazija desnog lobusa jetre--retka kongenitalna anomalija

Ectopic gallbladder, followed by other abnormalities in the anatomy of liver, biliary-pancreatic system, and portal vein is very rare congenital defect. The patient with left-sided gallbladder, complicated chronic calculous cholecystitis and hypoplasia of the right lobe of liver was presented. The diagnosis of this abnormality was confirmed intraoperatively. After cholecystectomy, postoperative course in the patient was uneventful.

[Personal experience in the diagnosis and treatment of hepatic echinococcosis]. / Nasa iskustva u dijagnostici i lecenju ehinokokoze jetre.

Human echinococcosis is endemic disease that occurs in some regions of Yugoslavia. It is caused by tapeworm Echinococcus granulosus, whose larva can develop cysts in liver and other organs. In the study were presented 119 patients with hepatic echinococcosis, 57 (47.9%) females of average age 41.4 years (9-80) and 62 (52.1%) males of average age 35.5 years (6.72). Primary echinococcosis was present in 75 (63%), and recurrent in 44 (37%) patients. Right lobe of liver was affected in 83 (69.7%), left in 17 (14.3%) and both lobes in 19 (16%) of cases. The complications of hepatic echinococcosis such as cyst infection, cholestasis, cyst rupture in biliary tract and liver fibrosis were observed in 20 (16.8%) patients. Indirect immunofluorescence antibody test was positive in 91 out of 119 (68.1%) patients, and hemagglutination inhibition test was positive in 56 out of 77 (72.7%) patients. Surgically were treated 57 (47.9%) patients, and 41 of them received antihelminithic drugs pre and/or postoperatively. Disease recurrence was observed in 4 (7%) patients. Percutaneous puncture and drainage of echinococcus cyst with simultaneous albendazole administration was performed in 12 (10%) patients. In three of them liver abscess was developed during drainage, and for that reason, two patients had to be surgically treated. Ten (83.3%) patients were completely cured. Medicamentous therapy as the only treatment was used in 31 (26.1%) patients, 9 patients received mebendazole, 19 received albendazole and 3 patients received praziquantel. The success was achieved in 10 (32.3%) patients. Out of the total number, 19 (16%) patients were not treated at all. Surgical removal of the cyst takes a leading place in the treatment of hepatic echinococcosis. However, in well-selected cases and in the patients with high surgical risk, antihelminithic therapy and percutaneous drainage of echinococcus cyst are of more significance.

[Use of fibrin glue prepared from recycled cryoprecipitate in experimental liver surgery]. / Primena fibrinskog lepka pripremljenog reciklaznom krioprecipitacijom u eksperimentnoj hirurgiji jetre.

Fibrin glue (FG) is a two-component biologic system with adhesive, sealant and topical hemostatic properties, containing fibrinogen (Fg), factor XIII (FXIII), fibronectin (Fn), thrombin, some antifibrinolytic agent if needed and ionized calcium. In this study, FG component 1 was prepared by recycling cryoprecipitation from single-donor plasma. The mean concentrations of Fg, FXIII and Fn were: 54.2 +/- 19.9 g/l, 8.03 +/- 2.3 IU/ml and 3103.1 +/- 148.91 mg/l, respectively. Horizontal tensile strength of FG was 1.076 +/- 0.18 N/cm2 in the average. Using a rat model, the efficacy of the FG-treatment in liver surgery was evaluated on the basis of the 24 hour survival ratio and hematological parameters of the experimental animals and control group. Survival of rats subjected to partial and total lobectomy and FG-treated was significantly (p < 0.001) higher than in FG-non-treated animals. Survival of animals subjected to liver incision was not significantly different, although the differences in hematological parameters were significant (p < 0.001 to p < 0.09) in favor of FG-treated animals. Our findings confirmed that high quality FG can be prepared by recycling cryoprecipitation from single-donor plasma--with sufficient yield of fibrinogen, FXIII and fibronectin and with the risk of disease transmission not greater than with the use of single unit of blood or plasma--which have efficient hemostatic and therapeutic properties.

[Complications after primary surgical management of war injuries of the colon and rectum]. / Komplikacije posle primarnog hirurskog zbrinjavanja ratnih povreda kolona i rektuma.

War injuries of colon and rectum are real challenge for war surgeons. The aim of the study was to point out the necessity of war surgical doctrine application, considering the risk factors in the choice of primary surgical procedure and surgeon's experience in managing this kind of injuries. Postoperative complications and indications for reoperations in 216 wounded after primary surgical management of colonic and rectal war injuries in the period 1991-1995 were retrospectively analyzed. A total of 183 wounded (84.7%) were primarily surgically managed in war hospitals. Combined injuries of colon and/or rectum or more than 2 abdominal organs were found in 197 wounded (91.2%), and 29.1% of wounded were also with the combined extra-abdominal injuries. Postoperative complications were observed in 58 wounded (26.8%) and were associated with the injury and primary management of colon and rectum, with septic complications in 87.9% of cases. Reoperation was performed in 44 wounded (75.8%) and the most frequent indications, such as diffuse peritonitis and stercoral fistula, were the sequlae of dehiscence of suture or anastomosis, overlooked lesions and secondary perforations (blast) of colon or rectum. Principle of complete diversion of stool/intestinal content was used in reoperation. Mortality rate in reoperated group was 20.4% (9 wounded). Cause of death was diffuse peritonitis followed by consecutive sepsis and MOFS. The significance of surgical war doctrine and risk factors in the choice of primary surgical procedures were emphasized.

[War injuries of the rectum and perineum]. / Ratne povrede rektuma i perineuma.

The treatment results of 35 wounded with rectal and perineal lesion, treated in the period from 1991 to 1993 were analyzed retrospectively. In military hospitals, 82% of wounded were primarily surgically managed. The majority (65.8%) was with injury of intraperitoneal rectal segment, 10 patients (28.5%) were with the lesion of extraperitoneal segment and 2 patients (5.7%) were with the injury of anal tract and perineum. The injuries were mostly inflected by the bullets of various calibers (42.6%). Multiple or combined injuries were found in 91.6% of wounded with rectal injury and 38.2% of them were managed within 6 hours after injury. Different surgical procedures, most frequently the lesion suture and proximal colostomy (n = 14) were primarily used. Postoperative complications occurred in 10 wounded (28.6%). In 3 wounded it was reintervened by Hartmann's procedure. Mortality rate was 8.5% (n = 3). Anorectal war injuries are the most severe of all the colonic injuries. To our experience and the unique war surgical doctrine in the primary treatment of rectum injury, the most important is to perform terminal colostomy, injury suture (always intraperitoneal, and extraperitoneal if possible, including the sphincter muscles approximation) and presacral drainage.

[The Miles-Thompson-Bricker method of total pelvic exenteration in malignant tumors of the rectum with urinary bladder infiltration]. / Totalna pelvicka egzenteracija po Miles-Thompson-Brickeru kod malignih tumora rektuma koji infiltruju mokracnu besiku.

The radical method of surgical treatment of malignant rectal neoplasms, total pelvic exenteration by Miles-Thompson-Bricker, was introduced in the Clinic for General and Vascular Surgery of Military Medical Academy in October, 1993, 45 years after the first operation of that kind in the world. This method of surgical treatment was indicated in 3% of our patients with malignant neoplasms of the distal part of rectum that infiltrated the urinary bladder. The life of patients with two stomas though not easy, was prolonged.

Occurrence of epidermal nerve endings in glabrous and hairy skin of the rat foot after sciatic nerve regeneration.

The occurrence and distribution of intraepidermal nerve endings in hairy and glabrous skin of the rat foot was examined in normal cases and three months after sciatic neurotomy/suture or a crush lesion. The nerve endings were visualized in cryostate sections with antibodies against protein gene product 9.5. Normal glabrous skin exhibited 23.3 endings/mm length. Neurotomy/suture cases had 6.1 endings/mm. In rats with nerve crush injury the occurrence was normal, but the intraepidermal nerve endings tended to be abnormally short and occurred mainly in the basal layer of the epidermis. In sections from hairy skin countings were not possible. Subjective evaluation indicated that the occurrence of dermal and epidermal axon profiles usually was very deficient after neurotomy/suture and essentially normal after crush. Skin samples from the contralateral side of operated animals showed a normal occurrence and distribution of nerve endings. We also noted that cases subjected to neurotomy/suture showed increased numbers of protein gene product 9.5-immunoreactive intraepidermal cells and an abnormally thin epidermis. We suggest that a highly deficient occurrence of intraepidermal nerve endings may be one factor behind the unsatisfactory restitution of the sensory function of the hand typically seen in patients subjected to neurotomy and suture.

Treatment of wounded in the combat zone.

Authors presented their own experiences in treating 735 wounded in high-intensity combat zones in the territories of former Yugoslavia during 1991 to 1992. The mobile field hospital with surgical crews was situated 5 to 10 km from the front line, and its basic task had been continuous triage, immediate resuscitation with vital surgical aid, as well as organization of adequate primary and secondary air evacuation. At the field hospital level, fresh wounds were explored according to principles of war surgery, and major surgical interventions were performed in 3.3% of the wounded. Patients with massive hematothorax were treated with autotransfusion. Mortality at this primary level, field hospital was 0.75% with primary immediate resuscitation and 1.9% with immediate evacuation. We concluded that immediate resuscitation with delayed transport had advantages, compared with fast evacuation of only the wounded.

Colon and rectal war injuries.

The results of retrospective analysis in the treatment of 189 wounded with colorectal lesions treated at the Military Medical Academy from July 1991 to December 1993 were presented. Primary surgical management was performed in 33 (17.5%) wounded. The others were transported into this hospital for further treatment after primary surgical management in war hospitals in the combat zone. Colorectal lesions (46.0%) were primarily inflicted by bullets of various calibers and high kinetic energy. The time interval between wounding and surgery was less than 6 hours for 39.6% of the wounded, whereas 80.1% were operated on within 12 hours. Multiple or combined colonic and rectal injuries were found in 91.5% of the wounded. Different surgical procedures were performed in accordance with the surgical war doctrine. Postoperative complications (colorectal cause) were found in 40 (21.2%) wounded. Reoperation was performed in 35 (18.5%) wounded. Total mortality rate was 10.1% (19 wounded).

Regeneration of putative sensory and sympathetic cutaneous nerve endings in the rat foot after sciatic nerve injury.

The present study examines the occurrence of calcitonin gene-related peptide-, substance P- and tyrosine hydroxylase-like immunoreactive profiles in glabrous and hairy foot skin from normal and nerve-injured rats. After neurotomy/suture, glabrous skin samples contain few calcitonin gene-related peptide-, substance P- and tyrosine hydroxylase-like immunoreactive profies. The number of calcitonin gene-related peptide- and substance P-like immunoreacive profiles in the epidermis is significantly subnormal. Hairy skin from these rats does also contain few calcitonin gene-related peptide-, substance P- and tyrosine hydroxylase-like immunoreactive profiles. In addition, the presence of epidermal calcitonin gene-related peptide-like imunoreactive profiles in glabrous skin is subnormal on the contralateral side. After nerve crush injury, the occurrence of calcitonin gene-related peptide-like, but not substance P-like, immunoreactive profiles in th epidermis of the glabrous skin is significantly subnormal. The occurrence of tyrosine hylase-like immnunoreactive fibres in relation to the digital artery is also subnormal. The occurrence in hairy skin of calcitonin gene-related peptide-like immunoreactive, substance P-like immunoreactive and tyrosine hydroxylase-like immunoreactive profiles is subnormal. In both skin types, the contralateral occurrence of such profiles is subjectively normal. These results show that the occurrence of calcitonin gene-related peptide-, substance P-, and tyrosine hydroxylase-like immunoreactive profiles in glabrous and hairy foot skin is clearly subnormal after neurotomy and suture and less abnormal after nerve crush. After neurotomy and suture the contralateral side is also affected.