Co-delivery of immunomodulators in biodegradable nanoparticles improves therapeutic efficacy of cancer vaccines.

To improve the efficacy of cancer vaccines we aimed to modulate the suppressive tumor microenvironment. In this study, the potential of intratumoral immune modulation with poly (I:C), Resiquimod (R848) and CCL20 (MIP3α) was explored. Biodegradable polymeric nanoparticles were used as delivery vehicles for slow and sustained release of these drugs in the tumor area and were combined with specific immunotherapy based on therapeutic peptide vaccination in two aggressive murine carcinoma and lymphoma tumor models. Whereas nanoparticle delivery of poly (I:C) or R848 improved therapeutic efficacy, the combination with MIP3α remarkably potentiated the cancer vaccine antitumor effects. The long-term survival increased to 75-100% and the progression free survival nearly doubled on mice with established large carcinoma tumors. The potent adjuvant effects were associated with lymphoid and myeloid population alterations in the tumor and tumor-draining lymph node. In addition to a significant influx of macrophages into the tumor, the phenotype of the suppressor tumor-associated macrophages shifted towards an acute inflammatory phenotype in the tumor-draining lymph node. Overall, these data show that therapeutic cancer vaccines can be potentiated by the combined nanoparticle mediated co-delivery of poly (I:C), R848 and MIP3α, which indicates that a more favorable milieu for cancer fighting immune cells is created for T cells induced by therapeutic cancer vaccines.

Combinatory therapy adopting nanoparticle-based cancer vaccination with immune checkpoint blockade for treatment of post-surgical tumor recurrences.

Cancer immunotherapy is emerging as a candidate treatment modality for treating post-surgical metastasis and recurrences. Despite the great promises with therapeutic cancer vaccines and checkpoint blocking antibodies in pre-clinical studies, response rates in the clinic still remain unsatisfactory. The evaluation of immunotherapy after surgery in patients could confront significant unexpected hurdles. Surgery itself tends to cause immune suppression, while wound healing factors also stimulate tumor cell outgrowth and metastasis. Regarding the marked changes in the post-surgical tumor microenvironment, one can anticipate that better tumor growth control is attainable by combining cancer vaccines with immune checkpoint blockade. However, it is important that vaccines and checkpoint blocking antibodies are delivered efficiently to their target cells, are released sustained and locally and do not induce cytotoxic effects. The generation of effective anti-tumor immunity and durable response rates could largely depend on these parameters. In the last decade, researchers spend tremendous effort in optimizing the delivery of immunotherapeutic compounds with the use of nanomedicine. Biocompatible nanoparticle based delivery systems demonstrated intriguing results with regard to specific immune cell activation, improved drug delivery, cell targeting, limiting off target toxicity and improving treatment outcome. It therefore makes sense, to speculate on the promises of combined cancer vaccination and immune checkpoint blocking immunotherapy with the aid of nanomedicine. A powerful nanoparticle combination immunotherapy conferring durable therapeutic benefit whilst leaving healthy tissue untouched represents the base for more efficient post-surgical cancer treatment.

The potential of multi-compound nanoparticles to bypass drug resistance in cancer.

PURPOSE: The therapeutic efficacy of conventional chemotherapy against several solid tumors is generally limited and this is often due to the development of resistance or poor delivery of the drugs to the tumor. Mechanisms of resistance may vary between cancer types. However, with current development of genetic analyses, imaging, and novel delivery systems, we may be able to characterize and bypass resistance, e.g., by inhibition of the right target at the tumor site. Therefore, combined drug treatments, where one drug will revert or obstruct the development of resistance and the other will concurrently kill the cancer cell, are rational solutions. However, drug exposure of one drug will defer greatly from the other due to their physicochemical properties. In this sense, multi-compound nanoparticles are an excellent modality to equalize drug exposure, i.e., one common physicochemical profile. In this review, we will discuss novel approaches that employ nanoparticle technology that addresses specific mechanisms of resistance in cancer. METHODS: The PubMed literature was consulted and reviewed. RESULTS: Nanoparticle technology is emerging as a dexterous solution that may address several forms of resistance in cancer. For instance, we discuss advances that address mechanisms of resistance with multi-compound nanoparticles which co-deliver chemotherapeutics with an anti-resistance agent. Promising anti-resistance agents are (1) targeted in vivo gene silencing methods aimed to disrupt key resistance gene expression or (2) protein kinase inhibitors to disrupt key resistance pathways or (3) efflux pumps inhibitors to limit drug cellular efflux.

The Potential of Nano-Vehicle Mediated Therapy in Vasculitis and Multiple Sclerosis.

The induction of immune tolerance towards self-antigens presents as a viable future strategy in the treatment of auto-immune diseases, including vasculitis and multiple sclerosis (MS). As specific targets are currently lacking for vasculitis due to incomplete understanding of the pathologies underlying this disease, current treatment options are based on modalities that induce general immune suppression. However, many immune suppressants used in the clinic are known to display wide biodistribution and are thus often accompanied by several adverse effects. Nano-vehicles (NVs) possess the ability to overcome such limitations by enabling more specific delivery of their content through modifications with targeting moieties. In this review, we describe the latest insights in the pathology of vasculitis that may function as potential targets for NV carrier systems, allowing more specific delivery of currently used immune suppressants. In addition, we describe the existing strategies to induce artificial immune tolerance and explore the feasibility of inducing regulatory T cell (Treg) mediated tolerance for MS, possibly mediated by NVs.

Combinatorial prospects of nano-targeted chemoimmunotherapy.

Despite the significant increase in our knowledge on cancer initiation and progression, and the development of novel cancer treatments, overall patient survival rates have thus far only marginally improved. However, it can be expected that lasting tumor control will be attainable for an increasing number of cancer patients in the foreseeable future, which is likely to be achieved by combining cancer chemotherapy with anticancer immunotherapy. A plethora of new cancer chemotherapy reagents are expected to become accessible to the clinic in the coming years which can then be used for efficient tumor debulking and aid in antigen exposure to the immune system. Durable remission and the eradication of micrometastases are likely to be achieved with specialized monoclonal antibodies and therapeutic cancer vaccines that modulate the immune system to overcome immunosuppression and kill distant cancer cells. Moreover, the method of drug delivery to tumors, stromal and immune cells is expected to shift largely from conventional 'free' drug molecules to encapsulated in targeted nano-vehicles, therapeutics often referred to or considered part of "nanomedicine". Several biocompatible nano-vehicles, such as metal-nanoparticles, biodegradable-nanoparticles, liposomes or dendrimers are potential candidates for targeted drug delivery but may also serve additional purposes. A dexterous combination of nanomedicine, cancer immunotherapy and chemotherapeutic engineering are likely to become the basis for new hope in the form of targeted cancer therapies that could attack tumors early in their development. One can envision nano-vehicles that would selectively deliver effective doses of chemotherapeutic agents to cancer cells while leaving healthy cells untouched. Furthermore, given that after chemotherapeutic treatment there often remains a limited number of chemo-resistant tumor cells, which go on to drive tumor progression, nano-vehicles could also be engineered to provoke an appropriate immune response to destroy these cells. Here, we discuss the potential of the combinatorial role of cancer chemotherapy, cancer immunotherapy and the prospective of nanotechnology for the targeted delivery of chemoimmunotherapeutic agents.

Significant lethality following liver resection in A20 heterozygous knockout mice uncovers a key role for A20 in liver regeneration.

Hepatic expression of A20, including in hepatocytes, increases in response to injury, inflammation and resection. This increase likely serves a hepatoprotective purpose. The characteristic unfettered liver inflammation and necrosis in A20 knockout mice established physiologic upregulation of A20 as integral to the anti-inflammatory and anti-apoptotic armamentarium of hepatocytes. However, the implication of physiologic upregulation of A20 in modulating hepatocytes' proliferative responses following liver resection remains controversial. To resolve the impact of A20 on hepatocyte proliferation and the liver's regenerative capacity, we examined whether decreased A20 expression, as in A20 heterozygous knockout mice, affects outcome following two-third partial hepatectomy. A20 heterozygous mice do not demonstrate a striking liver phenotype, indicating that their A20 expression levels are still sufficient to contain inflammation and cell death at baseline. However, usually benign partial hepatectomy provoked a staggering lethality (>40%) in these mice, uncovering an unsuspected phenotype. Heightened lethality in A20 heterozygous mice following partial hepatectomy resulted from impaired hepatocyte proliferation due to heightened levels of cyclin-dependent kinase inhibitor, p21, and deficient upregulation of cyclins D1, E and A, in the context of worsened liver steatosis. A20 heterozygous knockout minimally affected baseline liver transcriptome, mostly circadian rhythm genes. Nevertheless, this caused differential expression of >1000 genes post hepatectomy, hindering lipid metabolism, bile acid biosynthesis, insulin signaling and cell cycle, all critical cellular processes for liver regeneration. These results demonstrate that mere reduction of A20 levels causes worse outcome post hepatectomy than full knockout of bona fide liver pro-regenerative players such as IL-6, clearly ascertaining A20's primordial role in enabling liver regeneration. Clinical implications of these data are of utmost importance as they caution safety of extensive hepatectomy for donation or tumor in carriers of A20/TNFAIP3 single nucleotide polymorphisms alleles that decrease A20 expression or function, and prompt the development of A20-based liver pro-regenerative therapies.

Application of the mammalian glyceraldehyde-3-phosphate dehydrogenase gene for sample quality control in multiplex PCR for diagnosis of leishmaniasis

Leishmaniasis is a neglected disease endemic in five continents. It is a severe disease that may lead to death, and its early detection is important to avoid severe damage to affected individuals. Molecular methods to detect Leishmania are considered alternatives to overcome the limitations presented by conventional methods. The aim of this study was to develop multiplex PCR systems able to detect small amounts of target DNA of Leishmania infantum and Leishmania braziliensis, and the gene coding for glyceraldehyde-3-phosphate dehydrogenase (G3PD) in mammals, enabling quality evaluation of the sample simultaneously with detection of the specific target. The systems created for G3PD recognition were combined with detection systems for L. infantum and L. braziliensis to compose multiplex PCR systems for visceral (mVL) and cutaneous (mACL) leishmaniasis diagnosis. The multiplex PCR systems developed were assessed in blood samples from five different species of mammal reservoirs involved in the disease cycle in Brazil, and 96 and 52 human samples from patients with suspected visceral leishmaniasis (VL) and cutaneous leishmaniasis (ACL), respectively. Three G3PD detection systems were created (G3PD1, G3PD2 and G3PD3) with different product sizes, G3PD2 was chosen for the formation of multiplex PCR systems. The two multiplex PCR systems (mVL and mACL) were reproducible in all species evaluated. Results of test samples (sensitivity, specificity and efficiency) suggest its use in routine diagnosis, research activities in medicine and veterinary medicine. Additionally, the systems designed to detect the G3PD gene are capable of combining with other targets used for molecular diagnosis of infectious diseases. Concerning leishmaniasis, the multiplex PCR systems can be used in epidemiological studies for the detection of new and classic reservoirs, which may contribute to the reliability of results and development of actions to control the disease.(AU)

Comparison of real-time PCR and conventional PCR for detection of Leishmania (Leishmania) infantum infection: a mini-review

In recent years, the polymerase chain reaction (PCR) technique has significantly advanced towards expanding its use and versatility by working with quantitative real-time PCR (qPCR). Data from the literature show that both methods present interesting characteristics for the diagnosis of visceral leishmaniasis. The benefits of qPCR in relation to conventional PCR include speed, reproducibility and quantitative ability. In addition to operational advantages, qPCR is more sensitive and reproducible and may replace conventional PCR in diagnostic routines. Regarding visceral leishmaniasis, the possibility of deployment of real-time PCR in highly complex diagnoses (reference services) in endemic areas will facilitate a swift and safe return for patients. Moreover, the use of a technique that possesses elevated diagnostic sensitivity, and can monitor therapy and prevent relapses promotes broader prospects for the disease control.

Analysis of DNA polymerase activity in vitro using non-radioactive primer extension assay in an automated DNA sequencer.

Although different DNA polymerases have distinct functions and substrate affinities, their general mechanism of action is similar. Thus, they can all be studied using the same technical principle, the primer extension assay employing radioactive tags. Even though fluorescence has been used routinely for many years for DNA sequencing, it has not been used in the in vitro primer extension assay. The use of fluorescence labels has obvious advantages over radioactivity, including safety, speed and ease of manipulation. In the present study, we demonstrated the potential of non-radioactive in vitro primer extension for DNA polymerase studies. By using an M13 tag in the substrate, we can use the same fluorescent M13 primer to study different substrate sequences. This technique allows quantification of the DNA polymerase activity of the Klenow fragment using different templates and under different conditions with similar sensitivity to the radioactive assay.

Analysis of DNA polymerase activity in vitro using non-radioactive primer extension assay in an automated DNA sequencer

Although different DNA polymerases have distinct functions and substrate affinities, their general mechanism of action is similar. Thus, they can all be studied using the same technical principle, the primer extension assay employing radioactive tags. Even though fluorescence has been used routinely for many years for DNA sequencing, it has not been used in the in vitro primer extension assay. The use of fluorescence labels has obvious advantages over radioactivity, including safety, speed and ease of manipulation. In the present study, we demonstrated the potential of non-radioactive in vitro primer extension for DNA polymerase studies. By using an M13 tag in the substrate, we can use the same fluorescent M13 primer to study different substrate sequences. This technique allows quantification of the DNA polymerase activity of the Klenow fragment using different templates and under different conditions with similar sensitivity to the radioactive assay.

D-2-hydroxyglutaric acid inhibits creatine kinase activity from cardiac and skeletal muscle of young rats.

BACKGROUND: Tissue accumulation of high amounts of D-2-hydroxyglutaric acid (DGA) is the biochemical hallmark of the inherited neurometabolic disorder D-2-hydroxyglutaric aciduria (DHGA). Patients affected by this disease usually present hypotonia, muscular weakness, hypertrophy and cardiomyopathy, besides severe neurological findings. However, the underlying mechanisms of muscle injury in this disorder are virtually unknown. MATERIALS AND METHODS: In the present study we have evaluated the in vitro role of DGA, at concentrations ranging from 0.25 to 5.0 mM, on total, cytosolic and mitochondrial creatine kinase activities from skeletal and cardiac muscle of 30-day-old Wistar rats. We also tested the effects of various antioxidants on the effects elicited by DGA. RESULTS: We first verified that total creatine kinase (CK) activity from homogenates was significantly inhibited by DGA (22-24% inhibition) in skeletal and cardiac muscle, and that this activity was approximately threefold higher in skeletal muscle than in cardiac muscle. We also observed that CK activities from mitochondrial (Mi-CK) and cytosolic (Cy-CK) preparations from skeletal muscle and cardiac muscle were also inhibited (12-35% inhibition) by DGA at concentrations as low as 0.25 mm, with the effect being more pronounced in cardiac muscle preparations. Finally, we verified that the DGA-inhibitory effect was fully prevented by preincubation of the homogenates with reduced glutathione and cysteine, suggesting that this effect is possibly mediated by modification of essential thiol groups of the enzyme. Furthermore, alpha-tocopherol, melatonin and the inhibitor of nitric oxide synthase L-NAME were unable to prevent this effect, indicating that the most common reactive oxygen and nitrogen species were not involved in the inhibition of CK provoked by DGA. CONCLUSION: Considering the importance of creatine kinase activity for cellular energy homeostasis, our results suggest that inhibition of this enzyme by increased levels of DGA might be an important mechanism involved in the myopathy and cardiomyopathy of patients affected by DHGA.

Tubular cell lesion, albuminuria, and renal albumin handling in rats treated with adriamycin.

Several lines of evidence have suggested that renal handling of proteins in rats with several nephropathies may contribute to the tubulointerstitial damage observed in these animals. It has been suggested that proteins filtered by the glomeruli may be toxic for tubule cells. The aim of this study was to investigate the relationship between albuminuria and tubular lesions observed in rats during the first two weeks after treatment with adriamycin (AD). Thirty female Wistar rats were injected intravenously with adriamycin at the dose of 3.5 (17 rats) or 5mg/kg body weight (13 rats), and 7 were injected with 0.15 M NaCl (control group). Seven days later, we replaced drinking water with a 0.10 M sodium bicarbonate solution for 6 of the animals injected with 5 mg/kg adriamycin (group AD-B). Urine samples were collected before and 7 and 15 days after treatment to quantify albumin. The rats were killed 7 and 18 days after the injections, and the kidneys removed for immunohistochemical study. We observed a significant increase in urinary albumin excretion 15 days after AD injection (3.5 mg/kg), but not 7 days after AD. However, in the animals injected with 5.0 mg/kg AD (group AD-5) the increase in albuminuria was observed as early as on day 7. The immunohistochemical studies showed increased vimentin and albumin immunoreaction in the tubular cells of the renal cortex from the kidneys of rats injected with 3.5 mg/kg (group AD-3) only 18 days after treatment (p < 0.05), whereas in the animals treated with 5 mg/kg AD these immunohistochemical alterations were more intense. However, treatment with sodium bicarbonate attenuated the tubular lesions and reduced albumin reabsorption in adriamycin-treated rats. In conclusion, these experiments showed a relationship between albuminuria and tubular lesions in adriamycin-treated rats.

Heparin treatment reduces glomerular injury in rats with adriamycin-induced nephropathy but does not modify tubulointerstitial damage or the renal production of transforming growth factor-beta.

In this study we investigated the effect of heparin on renal injury and renal transforming growth factor-beta (TGF-beta) production in adriamycin (AD)-injected rats. Thirty-nine female Wistar rats were injected with AD (3.5 mg/kg body weight, i.v.) and 27 with 0.15 M NaCl solution (group C). Fifteen days later we started to inject heparin, 500 U/day, s.c., in 20 of the AD-injected animals (AD-H group). Three months after beginning treatment, urine samples were collected to quantify albumin, creatinine and TGF-beta. The rats were killed and the kidneys removed for histological, immunohistochemical, ELISA and RNA studies. All AD-injected animals showed structural renal changes (p < 0.05). However, the glomerular alterations were less intense in rats from group AD-H (p < 0.05). The percentage of glomerulosclerosis was 0.11 +/- 0.08 in group C, 14.7 +/- 12.8 in group AD (treated only with AD) and 3.42 +/- 2.3 in group AD-H. Renal cortex immunostaining for TGF-beta and mRNA content of this polypeptide was higher in both groups of animals injected with AD compared to controls (p < 0.05). These animals also presented a higher rate of urinary TGF-beta excretion (p < 0.05), which was 202 +/- 11 in group C, 1,103 +/- 580 in group AD and 1,564 +/- 328 pg/mg Ucreat in group AD-H. However, TGF-beta activity in the glomerular-conditioned media from the rats of group AD was higher than in the glomerular-conditioned media from the rats of group AD-H. In conclusion, treatment with heparin reduces glomerular damage in rats with AD-induced nephropathy but does not modify tubulointerstitial lesions or the renal production of TGF-beta.

Transforming growth factor beta activity in urine of patients with type 2 diabetes and diabetic nephropathy.

Diabetic nephropathy (DN) is characterized structurally by progressive mesangial deposition of extracellular matrix (ECM). Transforming growth factor-ss (TGF-ss) is considered to be one of the major cytokines involved in the regulation of ECM synthesis and degradation. Several studies suggest that an increase in urinary TGF-ss levels may reflect an enhanced production of this polypeptide by the kidney cells. We evaluated TGF-ss in occasional urine samples from 14 normal individuals and 23 patients with type 2 diabetes (13 with persistent proteinuria >500 mg/24 h, DN, 6 with microalbuminuria, DMMA, and 4 with normal urinary albumin excretion, DMN) by enzyme immunoassay. An increase in the rate of urinary TGF-ss excretion (pg/mg U Creat.) was observed in patients with DN (296.07 +/- 330.77) (P<0.001) compared to normal individuals (17.04 +/- 18.56) (Kruskal-Wallis nonparametric analysis of variance); however, this increase was not observed in patients with DMMA (25.13 +/- 11.30) or in DMN (18.16 +/- 11.82). There was a positive correlation between the rate of urinary TGF-ss excretion and proteinuria (r = 0.70, alpha = 0.05) (Pearson's analysis), one of the parameters of disease progression.

Undernutrition decreases serine palmitoyltransferase activity in developing rat hypothalamus.

BACKGROUND/AIMS: Undernutrition reduces the hypothalamic ganglioside concentration. This may be attributed to some modifications in the contents of precursors of sphingolipid biosynthesis in undernourished rats. The present study evaluated the serine palmitoyl transferase activity (SPT; EC 2.3.1.50) during the development of the rat hypothalamus. This work also shows the L-[3-(14)C]serine metabolic labeling of hypothalamic sphingolipids in normal and undernourished rats at weaning. METHODS: The SPT activity was determined in microsomal fractions obtained from the hypothalamus of normal rats (diet: 25% protein) and pre- and postnatally undernourished rats (diet: 8% protein since pregnancy) at 21 days of gestational age and at 7, 14, and 21 days of postnatal life. RESULTS: The enzymatic activity was lower in the hypothalamus of undernourished than in the hypothalamus of control rats since the 7th postnatal day. Incorporation of the precursor L-[3-(14)C]serine into sphingolipid fraction was lower in the hypothalamus of undernourished rats than in the hypothalamus of control rats on the 21st postnatal day which coincided with the age of the highest difference in SPT activity between normal and undernourished rats. CONCLUSION: These results indicate that undernutrition reduces the biosynthesis of the main sphingolipids during the period of brain growth spurt.

In vitro inhibition of Na+,K(+)-ATPase activity from rat cerebral cortex by guanidino compounds accumulating in hyperargininemia.

Hyperargininemia is a metabolic disorder biochemically characterized by tissue accumulation of arginine (Arg) and other guanidino compounds (GC). Convulsions, lethargy and psychomotor delay are predominant clinical features of this disease. Considering that some GC are epileptogenic and cause a decrease in membrane fluidity and that Na+,K(+)-ATPase, a membrane-bound enzyme, is essential for cellular excitability and is decreased in experimental and human epilepsy, in the present study we determined the in vitro effects of Arg, N-acetylarginine (NAA), argininic acid (AA) and homoarginine (HA) on the activity of Na+,K(+)-ATPase in the synaptic plasma membrane from cerebral cortex of young rats in the hope to identify a possible mechanism for the brain damage in hyperargininemia. The results showed that all GC tested, except Arg, significantly inhibited Na+,K(+)-ATPase activity at concentrations similar to those observed in plasma and CSF of patients with hyperargininemia. In addition, competition between NAA, AA and HA for the binding to the enzyme was observed, suggesting a common binding site for the GC. It is therefore possible that the inhibitory effect of GC on Na+,K(+)-ATPase may be related to the brain dysfunction observed in hyperargininemia.

Chronic administration of propionic acid reduces ganglioside N-acetylneuraminic acid concentration in cerebellum of young rats.

Elevated levels of propionate comparable to those of human propionic acidaemia were achieved in the blood of young rats by injecting subcutaneously buffered propionic acid (PPA) twice a day at 8-h intervals from the 6th to the 28th day of life. A matched group of animals (controls) was treated with the same volumes of saline. The animals were weighed and sacrificed by decapitation at 28, 35 or 60 days of age. Cerebellum and cerebrum were weighed and their protein and ganglioside N-acetylneuraminic acid (G-NeuAc) contents determined. Body, cerebral and cerebellar weights were similar in both groups, suggesting that PPA per se neither alters the appetite of the rats nor causes malnutrition. Brain protein concentration was also not affected by chronic administration of PPA, in contrast to G-NeuAc concentration which was significantly reduced in the cerebellum. Since ganglioside concentration is closely related to the dendritic surface and indirectly reflects synaptogenesis, our results of an important ganglioside deficit in the brain of PPA-treated animals may be related to the neurologic dysfunction characteristic of propionic acidaemic patients.

Transforming growth factor-beta production during the development of renal fibrosis in rats with subtotal renal ablation.

The histologic changes observed in the remnant kidney model include progressive mesangial expansion with collapse of capillary lumina, interstitial fibrosis and mononuclear cellular infiltration. Transforming growth factor-beta (TGF-beta 1) is an important regulator of extracellular matrix formation. The purpose of this study was to investigate the production and distribution of TGF-beta 1 in the kidney during the development of glomerulosclerosis and renal fibrosis in rats with subtotal renal ablation. Eighty-two female Wistar rats weighing 180-220 g were divided into two groups: 49 rats were subjected to 5/6 renal ablation and 33 to sham operation. Urinary albumin excretion, blood pressure and glomerular filtration rate (GFR) were evaluated after the surgical procedure. We also performed histology and immunohistochemistry and determined mRNA for TGF-beta 1 in the kidneys of these rats 8, 15, 30 and 90 days after operation. The results showed progressively higher immunohistochemical TGF-beta 1 staining in rats with subtotal renal ablation. Cortical renal content of TGF-beta 1 mRNA was also higher in these animals and peaked at day 15. The existence of a temporal association between glomerulosclerosis, interstitial fibrosis and intense mononuclear cellular infiltration on the one hand and higher immunohistochemical TGF-beta 1 staining in the renal cortex on the other show that this polypeptide may contribute to the development of renal fibrosis in this model.

Multihormonal response to CRH in a patient with Cushing syndrome and a pituitary adenoma producing ACTH and GH.

Preoperative bilateral and simultaneous catheterization of the inferior petrosal sinuses was performed in a patient with Cushing's syndrome. This procedure revealed parallel intersinus gradients for ACTH, PRL, GH and a response of all these hormones to CRH. The patient had transphenoidal surgery and a soft white-yellowish mass occupying the whole sella conditioned a total hypophysectomy. Morphologic studies and immunostaining techniques revealed a rare mixed pituitary adenoma of ACTH and GH containing cells and failed to demonstrate PRL immunoreactivity in the tumor cells. These results are of clinical and speculative interest. Whether GH and PRL secretion in response to CRH stems from the tumor or reflect hormone release by non-tumor cells via a paracrine effect remains to be defined.