Physiological effects of a single chest physiotherapy session in mechanically ventilated and extubated preterm neonates.

OBJECTIVE: To assess the changes on various physiological cardio-respiratory parameters with a single chest physiotherapy session in mechanically ventilated and extubated preterm neonates with respiratory distress syndrome. STUDY DESIGN: This is a prospective observational study in a neonatal intensive care unit setting. Sixty preterm neonates with respiratory distress syndrome, thirty mechanically ventilated and thirty extubated preterm neonates requiring chest physiotherapy were enrolled in the study. Parameters like heart rate (HR), respiratory rate (RR), Silverman Anderson score (SA score in extubated), oxygen saturation (SpO2) and auscultation findings were noted just before, immediately after chest physiotherapy but before suctioning, immediately after suctioning and after 5 minutes of the session. RESULTS: The mean age of neonates was 9.55±5.86 days and mean birth weight was 1550±511.5 g. As there was no significant difference in the change in parameters on intergroup comparison, further analysis was done considering two groups together (n = 60) except for SA score. As SA score was measured only in extubated neonates. HR did not change significantly during chest physiotherapy compared to the baseline but significantly decreased after 15 minutes (p = 0.01). RR and SA score significantly increased after suctioning (p = 0.014) but reduced after 15 minutes (p = <0.0001). SpO2 significantly reduced post-suctioning compared to the baseline and increased after positioning and 15 minutes of chest physiotherapy (p = <0.0001). Clinically, there was a reduction in HR, RR and SA score with an improvement in SpO2. This signifies that chest physiotherapy may help facilitate the overall well-being of a fragile preterm neonate. Lung auscultation finding suggests that after suctioning, there was a significant reduction in crepitation (p = 0.0000) but significant increase in crepitation after 15 minutes (p = <0.01), suggesting the importance of around-the-clock chest physiotherapy. CONCLUSION: Chest physiotherapy is safe in preterm neonates. Suctioning causes significant cardio-respiratory parameter changes, but within normal physiological range. Thus, chest physiotherapy should be performed with continuous monitoring only when indicated and not as a routine procedure. More research is needed to study the long term effects of chest physical therapy.

The chronological appearance of flat colonic neoplasias in rats.

Following weekly s.c. injections of the colonotropic carcinogen 1,2-dimethylhydrazine (DMH), we investigated the occurrence of flat and protruding neoplasias in the colon of rats at various time intervals. Forty-seven DMH-treated rats were sacrificed at the 13th, 15th, 19th, 21st and 22nd weeks. A total of 88 tumors evolved in 35 of the 47 DMH-treated rats. The number of neoplasias/animal was 0.3 at week 13, 1.2 at week 15, 2.0 at week 19, 2.5 at week 21 and 4.0 at week 22. In the right colon, although the percent of flat adenomas was lower than of protruding adenomas, the percent of flat carcinomas was significantly higher than of protruding carcinomas, indicating that flat adenomas progress more rapidly to invasive carcinoma than protruding adenomas in the right colon. The opposite was recorded in the left colon where the percent of protruding adenomas was lower than of flat adenomas, but the percent of protruding carcinomas was higher than of flat carcinomas. During the last experimental week as many as 63% of the protruding carcinomas occurred but only 25% of the flat carcinomas. These experimental results seem to substantiate previous observations in humans suggesting that, in the colonic mucosa, flat and protruding adenomas follow different pathways of neoplastic transformation.

Clinical effects of monoclonal antibody 17-1A combined with granulocyte/macrophage-colony-stimulating factor and interleukin-2 for treatment of patients with advanced colorectal carcinoma.

Granulocyte/macrophage-colony-stimulating factor (GM-CSF) has previously been indicated to enhance the therapeutic effect of the anti-colorectal carcinoma mAb17-1A as well as to augment in vivo immune effector functions. In vitro interleukin-2 (IL-2) augmented GM-CSF-induced antibody-dependent cellular cytotoxicity, a mechanism considered to be of significance for the therapeutic effect of mAb. A treatment regimen was elaborated that combined mAb17-1A (400 mg at day 3 of a 10-day treatment cycle) with the simultaneous administration of GM-CSF (250 microgram/m(2) once daily) and IL-2 (2.4 x 10(6) U/m(2) twice daily) for 10 days. The treatment cycle was repeated once a month. Twenty patients with advanced colorectal carcinoma were included in the study. One patient obtained a partial remission and 2 patients stable disease for 7 and 4 months respectively. The median survival time from the start of mAb therapy was 8 months. Owing to allergic reactions, the planned mAb17-1A dose had to be reduced by repeated infusions. At the fourth treatment cycle only 25% received the planned mAb dose. In 3 patients the GM-CSF and IL-2 dose was reduced because of side-effects. The subjective tolerability of the treatment was considered good or acceptable in more than 80% of the patients. The increment in white blood cell subsets induced by the cytokines decreased by increasing number of courses. This particular regimen did not augment the therapeutic effect of mAb17-1A anticipated from in vitro data but rather hampered the clinical effect of the antibody. The reason for this is not clear but a possibility might be the induction of immune suppression in vivo resulting from an impaired human anti-(mouse Ab) and anti-idiotypic antibody response as well as antibody-dependent cellular cytotoxicity, on the basis of a comparison of mAb17-1A/GM-CSF/IL-2- and mAb17-1A/GM-CSF-treated patients.

Non-polypoid adenomas of the colon are associated with subjacent lymphoid nodules. An experimental study in rats.

BACKGROUND: Subjacent lymphoid nodules (SLNs) have been found in 38% of non-polypoid colonic adenomas in humans. In the present work the presence of SLNs in experimentally induced colonic adenomas was investigated in rats. METHODS: 1,2-Dimethylhydrazine was injected subcutaneously in 290 Sprague-Dawley rats for 27 weeks. RESULTS: An SLN was present in 28.6% of the 84 adenomas, in 8.4% of the 119 adenocarcinomas, and in 9.7% of the 31 small carcinomas without remnant adenomatous tissue. An SLN was found in 35.6% of the 59 non-polypoid neoplasias but only in 9.1% of the 175 polypoid (that is, exophytic) neoplasias. When only adenomas were considered, SLNs were present in 50.0% of the 34 non-polypoid adenomas but only in 14.0% of the 50 polypoid adenomas. CONCLUSIONS: Non-polypoid colonic adenomas evolve preferentially from the minimal fraction of the colonic mucosa that overlays the few existing lymphoid nodules in rats.

Augmentation of the immune response with granulocyte-macrophage colony-stimulating factor and other hematopoietic growth factors.

Granulocyte-macrophage colony-stimulating factor is by far the most widely used hematopoietic growth factor to augment immune responses. At present, the best secured effect is as an adjuvant cytokine for vaccination. Granulocyte-macrophage colony-stimulating factor can be delivered as gene-transduced tumor cells, as plasmid DNA, or as the soluble free granulocyte-macrophage colony-stimulating factor protein. Granulocyte-macrophage colony-stimulating factor must be present at the same site as the vaccine component. Granulocyte-macrophage colony-stimulating factor may also augment the effect of therapeutic monoclonal antibodies by enhancing various effector functions such as antibody-dependent cellular cytotoxicity and amplifying an idiotypic network response (i.e., antitumor immunity). It may also be advantageous to combine granulocyte colony-stimulating factor with monoclonal antibodies (neutrophil and monocyte antibody-dependent cellular cytotoxicity) for tumor therapy. However, these growth factors might also induce immune suppression, which may hamper the contemplated effect of the growth factor. It is urgently warranted to better understand these dual effects on the immune system so that we can find optimal uses for the growth factors in various clinical settings.

Immunopathology of metastases in patients of colorectal carcinoma treated with monoclonal antibody 17-1A and granulocyte macrophage colony-stimulating factor.

Twenty patients with metastatic colorectal carcinoma were treated with a single infusion (400 mg) of a mouse monoclonal antibody (IgG2a) against the tumor-associated antigen CO 17-1A and with a daily injection of granulocyte macrophage colony-stimulating factor (GM-CSF) for 10 days. The cycle was repeated every month. Metastases from 5 of the 20 patients biopsied on days 1 and 10 of the first two treatment cycles were studied by immunohistochemistry. During treatment, neutrophils, monocytes, and T lymphocytes increased concordantly in the tumor as in the blood of the individual patient. Macrophages (CD68) and CD8+ T cells infiltrated the tumor glands and displayed TIA-1-reactive cytotoxic granules. Neutrophils were seen mainly in areas of necrosis. Activated (HLA-DR+) CD4+ T cells were usually abundant in the stroma. During treatment, few natural killer cells were found in the tumor, contrary to the marked increase seen in blood. Our observations indicate that GM-CSF markedly recruited activated, tumor-infiltrating leukocytes, possibly representing antibody-dependent cellular cytotoxicity and cytotoxic T effector cells. The notion that combined antibody and GM-CSF therapy may also promote a T-cell antitumor response is further supported and advocated by our findings. The study lends further support to combining GM-CSF with monoclonal antibody-based therapy.

Retrieval of leukocyte antigens in paraffin-embedded rat tissues.

We compared the immunohistochemical reactivity of various rat leukocyte antigens in frozen and paraffin-embedded thymus, spleen, abdominal lymph node, liver, and brain tissues of healthy Sprague-Dawley rats, fixed in various fixatives. Immune reactivity after fixation in Methacarn was superior to that of 4% neutral buffered formalin, a mercury-based fixative (B-5), or Carnoy. Microwave (MW) antigen retrieval (AR) enhanced antigen reactivity. Ten of the 11 leukocyte antigens studied could be retrieved in Methacarn-fixed, paraffin-embedded sections with a reactivity comparable to that obtained on frozen sections.

Subcutaneous interleukin-2 and alpha-interferon in advanced colorectal carcinoma. A phase II study.

Subcutaneous administration of low doses of recombinant interleukin-2 (IL-2) and interferon-alpha (IFN-alpha) on an out-patient basis has been reported not to significantly compromise the response frequency compared to intravenous IL-2 in patients with renal cell carcinoma and melanoma. As part of an ongoing program to develop a biotherapeutic concept in patients with colorectal carcinoma (CRC) we studied the clinical effects of such a regimen in 15 patients with metastatic CRC. The daily dose of IL-2 varied between 4.8-14.4 x 10(6) U/m2 and of IFN-alpha between 3-6 x 10(6) U/m2. The cycle length was 6 weeks. The course was repeated every 8 weeks until disease progression. Maximum 4 cycles were administered. Maintenance therapy was given to responding patients once a week every month. No patient showed a major response (CR or PR). Six patients had a stable disease ranging from 3 months to 18 months with a median duration time of 5 months. The median survival of all patients was 13 months. The main adverse reactions were fever, chills, anorexia and shortness of breath. No treatment related deaths occurred. 6/14 patients developed abnormal concentration of serum levels of thyroid hormones. It is concluded that the present treatment schedule using IL-2 and IFN-alpha in advanced CRC seemed not to be of clinical benefit.

Intracerebral inflammatory response to experimental brain contusion.

The inflammatory reaction following experimental brain contusion was studied by immunohistochemistry in 22 rats during the first 16 days after trauma. An inflammatory mononuclear cell response was evident on day 2, with a maximum on days 5-6 and signs remained still 16 days after the trauma. The time course of the cellular infiltration adjacent to the lesion correlated with blood brain barrier dysfunction in the contralateral side of the traumatized hemisphere. The cellular infiltrate comprised NK cells, T-helper cells and T-cytotoxic/suppressor cells as well as monocytes/macrophages. Most of the macrophages appeared to be activated by T-cells. Surprisingly, polymorphonuclear cells appeared less engaged than mononuclear cells in the inflammation. The demonstration of immunocompetent cells and the induction of MHC-1 and MHC-II antigen provides a substrate for inflammatory reactions similar to those that cause neurological damage in inflammatory diseases such as viral infections, multiple sclerosis and experimental allergic encephalitis. Our observations indicate that the role of the inflammatory reactions may have a role, hitherto neglected, in the pathogenesis of secondary traumatic brain injury.

Flat adenoma-adenocarcinoma sequence in the colon of rats.

PURPOSE: As there is an increased awareness of the existence of a "flat adenoma-adenocarcinoma sequence" in the colonic mucosa of human subjects, the aims of the study were to assess whether flat colonic adenocarcinomas in rats are also preceded by flat adenomas, as is reported in humans, and to determine the frequency of flat lesions compared with exophytic lesions in the colon of rats. METHOD: The colonotropic carcinogen 1,2-dimethylhydrazine was injected subcutaneously in 300 Sprague-Dawley rats for 27 weeks. RESULTS: A total of 358 tumors developed in 278 of the 300 rats. Of the 60 adenomas found at histology, 25 percent were flat adenomas. Of the 298 adenocarcinomas, 12.7 percent had originated in a flat adenoma. Of the 180 colonic neoplasias (adenomas or adenocarcinomas), 29.4 percent were flat neoplasias (flat adenomas or adenocarcinomas arising in a flat adenoma), and the remaining 70.6 percent were exophytic neoplasias (tubulo or villous adenomas or adenocarcinomas arising in exophytic adenomas). From the 298 colonic adenocarcinomas, 1 was a intramucosal adenocarcinoma, 87 were overt adenocarcinomas, and 90 were lymphoid-associated carcinomas; in those 298 adenocarcinomas, no preneoplastic lesion could be recorded. In 208 animals, biopsies were taken from macroscopically visible colonic lesions, and, in the remaining 70 animals, the entire colon was processed for histologic examination. Flat adenomas were found in 3.8 percent of the 208 biopsy specimens and in 10 percent of the 70 colectomy specimens. Further, of the 40 adenomas found in biopsy specimens, 20 percent were flat adenomas, and, of the 20 adenomas found in colectomy specimens, 35 percent were flat adenomas. CONCLUSIONS: The study reported herein indicates the existence of a "flat adenoma-adenocarcinoma sequence" in the colonic mucosa of Sprague-Dawley rats. The flat lesions of the colon constituted approximately one-third of the total neoplastic lesions seen in the rat following injections of 1,2-dimethylhydrazine. More flat adenomas were detected at histologic examination of the entire colon than in biopsies obtained from the macroscopically visible colonic lesions. Consequently, flat adenomas may be overlooked by naked-eye examination.

Ear tumours induced by experimental carcinogenesis in the rat: excision prevents early death.

1,2-dimethylhydrazine (DMH) is widely used to induce colorectal tumours in rodents. Some of the animals develop ear as well as colorectal tumours. Rats with large, ulcerated ear tumours are usually sacrificed before the completion of the experiment. In this experiment, fourty-six male Spraque-Dawley rats were injected with 1,2-dimethylhydrazine (21 mg/kg body weight) once a week for 27 weeks to study the histogenesis of colorectal carcinoma. Thirty-six developed ear tumours. Fourteen of the 36 tumours were larger than 2 cm in diameter. These developed between 20-26 weeks and were surgically excised 1-5 weeks later. Four rats died postoperatively. The surgical removal of large ear tumours permitted the completion of the large bowel experiment on schedule (i.e. 27 weeks) in 10 (28%) of the 36 rats with ear tumours.

Histogenesis of small colonic adenocarcinomas.

It has been claimed that in order to identify the type of mucosal lesion which precedes colonic adenocarcinoma only those tumours measuring < or = 1 mm in diameter should be considered. The review of the English literature indicates that only 35 colorectal adenocarcinomas measuring < or = 1 cm in diameter have been so far reported. From the three cases of colonic adenocarcinoma measuring 8 mm or less in diameter presented here, one adenocarcinoma originated in a flat adenoma, the second in an exophytic tubular adenoma, and the third showed no adenomatous component. It would thus appear that colonic adenocarcinomas can originate not only from exophytic adenomas or from flat adenomas but also from apparently nonadenomatous flat mucosa.

Autologous transplanted colonic carcinoma in Sprague-Dawley rats.

Thirty-five adult male Sprague-Dawley rats received weekly subcutaneous injections of 1,2-dimethylhydrazine (DMH) for 12 weeks. At the end of this period, 31 rats had developed colonic carcinomas (CC). A fragment (1 mm3) of each CC was autotransplanted into the subcapsular space of the left kidney of the respective rats. After an additional 3 weeks, the rats were sacrificed. The left kidney in 20 of the 31 rats (65%) demonstrated tumors at the site of transplantation. The transplanted tumors demonstrated markers of viability such as a PCNA positive cells, mitotic figures, mucin secretion, neo-vascularization, invasion of the adjacent kidney tissue and infiltration of mononuclear cells. The frequency of tumor cells exhibiting the tumor associated antigens CO17-1A, GA73-3, BR55-2, GICA 19-9 was similar or higher in most of the autotransplants when compared to the donor tumor. Since MAb17-1A is being used in immunotherapy of metastatic CC in humans, the present model may offer a feasible method to assess various biotherapeutic approaches of metastatic CC based on the use of MAb17-17A.

Normal colon of Sprague-Dawley rats. An immunohistochemical study.

The patterns of expression of the human-tumor-associated antigens, CO17-1A, GA73-3, BR55-2, GICA19-9, CA50 and carcino-embryonic antigen (CEA) were studied in the normal colonic mucosa (the last three also in the serum) of Sprague-Dawley rats. Four immunohistochemically different segments were identified: caecum, ascending colon, transverse colon and descending colon. The immunohistochemical reactions of the cells at the lower part of the crypt were essential for the distinction of the four segments. In the caecum, the MAbs 17-1A, 73-3 and 19-9 stained the glycocalyx of the cells of the lower part of the crypts and the Golgi apparatus of the intercalated cells (IC). MAb55-2 stained very weakly the goblet-like cells (GLC) of the lower part of the crypt of transverse colon, in addition to a nearly complete lack of reaction in the upper part of the crypts. In the ascending colon, the lower part of the crypts showed a characteristic diffuse staining of the intercalated cells with MAb55-2. The perinuclear and mucosal staining observed in the GLC of the transverse colon with MAbs 17-1A, 73-3 and 19-9 as against the supranuclear and Golgi zone staining observed in the GLC/goblet cells (GC)/columnar cells (CC) of the lower part of crypts of the descending colon with the same MAbs, distinguished the former segment from the latter. The IC demonstrated by immunohistochemistry in the lower parts of the crypts of caecum and ascending colon appear to correspond to the replicating cells of the colonic crypts.

Tumor-associated antigens common to humans and chemically induced colonic tumors of the rat.

The expression of human tumor-associated antigens CO17-1A, GA73-3, BR55-2, GICA 19-9, and CA50 and of carcinoembryonic antigen was immunohistochemically studied in the colonic mucosa of 70 Sprague-Dawley rats. Fifty were treated with 1,2-dimethylhydrazine (DMH) (with EDTA as a vehicle), ten were treated with EDTA only, and ten were untreated normal rats. The tumors were histogenetically divided as: (a) adenocarcinomas arising from villous adenomas; (b) adenocarcinomas arising from lymphoid-associated mucosa (LAM); and (c) adenocarcinomas arising in flat mucosa. Of 44 colonic adenocarcinomas, BR55-2 was expressed in 41 tumors, CO17-1A in 40 tumors, GA73-3 in 38 tumors, and GICA 19-9 in 38 tumors. CA50 and carcinoembryonic antigen were not expressed in the tumors. The highest antigenic expression (number of cells) was observed in adenocarcinomas arising in villous adenomas and the lowest in those arising in flat mucosa. Adenocarcinomas arising in LAM had an intermediate expression. The expression of these antigens had no correlation to the localization of the tumor and to the differentiation. The expression of these antigens was similar in the non-lymphoid-associated normal colonic mucosa of the untreated, EDTA-treated, and DMH-treated rats. In DMH-treated rats, LAM demonstrated increased expression (number of cells) and increased staining intensity of these tumor-associated antigens. In six of the 50 DMH-treated rats, only LAM expressed carcinoembryonic antigen. CA50 was not expressed in the normal colon of untreated, of EDTA-treated, and of DMH-treated rats, nor was it in DMH-induced tumors. None of the tumor-associated antigens (GICA 19-9 and CA50 and carcinoembryonic antigen) was detected in serum. It is concluded that this animal model would be of value in the preclinical evaluations of monoclonal antibodies for therapy in humans.

The tumor-associated antigens BR55-2, GA73-3 and GICA 19-9 in normal and corresponding neoplastic human tissues, especially gastrointestinal tissues.

Immunohistochemical analysis with a monoclonal antibody, anti-BR55-2, was carried out on 163 tumors with their adjacent normal tissues and on 51 normal tissues from various organs by the ABC method. The expression of the antigen BR55-2 was compared with the expression of the colorectal carcinoma (CRC) associated antigens, GA73-3 and GICA19-9. BR55-2 was expressed in most normal epithelial tissues, whereas in the colon it seems to be exclusively a tumor-associated antigen. MAb55-2 might be of value in studying dysplastic lesions of the colon and in assessing the depth of invasion of CRC. In CRC, the expression of BR55-2 was complementary to that of GA73-3. MAb55-2 may therefore be of value in immunotherapy of CRC.